AlgiMatrix™-Based 3D Cell Culture System as an In Vitro Tumor Model: An Important Tool in Cancer Research.

PubMed

Godugu, Chandraiah; Singh, Mandip

2016-01-01

Routinely used two-dimensional cell culture-based models often fail while translating the observations into in vivo models. This setback is more common in cancer research, due to several reasons. The extracellular matrix and cell-to-cell interactions are not present in two-dimensional (2D) cell culture models. Diffusion of drug molecules into cancer cells is hindered by barriers of extracellular components in in vivo conditions, these barriers are absent in 2D cell culture models. To better mimic or simulate the in vivo conditions present in tumors, the current study used the alginate based three-dimensional cell culture (AlgiMatrix™) model, which resembles close to the in vivo tumor models. The current study explains the detailed protocols involved in AlgiMatrix™ based in vitro non-small-cell lung cancer (NSCLC) models. The suitability of this model was studied by evaluating, cytotoxicity, apoptosis, and penetration of nanoparticles into the in vitro tumor spheroids. This study also demonstrated the effect of EphA2 receptor targeted docetaxel-loaded nanoparticles on MDA-MB-468 TNBC cell lines. The methods section is subdivided into three subsections such as (1) preparation of AlgiMatrix™-based 3D in vitro tumor models and cytotoxicity assays, (2) free drug and nanoparticle uptake into spheroid studies, and (3) western blot, IHC, and RT-PCR studies.

A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

PubMed

Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

2015-09-01

A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.

The evolution of chicken stem cell culture methods.

PubMed

Farzaneh, M; Attari, F; Mozdziak, P E; Khoshnam, S E

2017-12-01

1. The avian embryo is an excellent model for studying embryology and the production of pharmaceutical proteins in transgenic chickens. Furthermore, chicken stem cells have the potential for proliferation and differentiation and emerged as an attractive tool for various cell-based technologies. 2. The objective of these studies is the derivation and culture of these stem cells is the production of transgenic birds for recombinant biomaterials and vaccine manufacture, drug and cytotoxicity testing, as well as to gain insight into basic science, including cell tracking. 3. Despite similarities among the established chicken stem cell lines, fundamental differences have been reported between their culture conditions and applications. Recent conventional protocols used for expansion and culture of chicken stem cells mostly depend on feeder cells, serum-containing media and static culture. 4. Utilising chicken stem cells for generation of cell-based transgenic birds and a variety of vaccines requires large-scale cell production. However, scaling up the conventional adherent chicken stem cells is challenging and labour intensive. Development of a suspension cell culture process for chicken embryonic stem cells (cESCs), chicken primordial germ cells (PGCs) and chicken induced pluripotent stem cells (ciPSCs) will be an important advance for increasing the growth kinetics of these cells. 6. This review describes various approaches and suggestions to achieve optimal cell growth for defined chicken stem cells cultures and use in future manufacturing applications.

Perfusion enhanced polydimethylsiloxane based scaffold cell culturing system for multi-well drug screening platform.

PubMed

Tania, Marshella; Hsu, Myat Noe; Png, Si Ning; Leo, Hwa Liang; Toh, Guoyang William; Birgersson, Erik

2014-01-01

Conventional two-dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver-specific functions and their cuboidal morphology over longer term culture. In this study, we present a three-dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver-specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N-Acetyl-Para-Amino-Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold-bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS-based scaffold system provides a more conducive microenvironment with better cell-to-cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing. © 2014 American Institute of Chemical Engineers.

Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture

NASA Astrophysics Data System (ADS)

Gangadharan, Rajan

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions.

Glutathione-Triggered Formation of a Fmoc-Protected Short Peptide-Based Supramolecular Hydrogel

PubMed Central

Shi, Yang; Wang, Jingyu; Wang, Huaimin; Hu, Yanhui; Chen, Xuemei; Yang, Zhimou

2014-01-01

A biocompatible method of glutathione (GSH) catalyzed disulfide bond reduction was used to form Fmoc-short peptide-based supramolecular hydrogels. The hydrogels could form in both buffer solution and cell culture medium containing 10% of Fetal Bovine Serum (FBS) within minutes. The hydrogel was characterized by rheology, transmission electron microscopy, and fluorescence emission spectra. Their potential in three dimensional (3D) cell culture was evaluated and the results indicated that the gel with a low concentration of the peptide (0.1 wt%) was suitable for 3D cell culture of 3T3 cells. This study provides an alternative candidate of supramolecular hydrogel for 3D cell culture and cell delivery. PMID:25222132

Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells.

PubMed

Mizukami, Amanda; Fernandes-Platzgummer, Ana; Carmelo, Joana G; Swiech, Kamilla; Covas, Dimas T; Cabral, Joaquim M S; da Silva, Cláudia L

2016-08-01

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering three dimensional micro nerve tissue using postnatal stem cells from human dental apical papilla.

PubMed

Kim, Byung-Chul; Jun, Sung-Min; Kim, So Yeon; Kwon, Yong-Dae; Choe, Sung Chul; Kim, Eun-Chul; Lee, Jae-Hyung; Kim, Jinseok; Suh, Jun-Kyo Francis; Hwang, Yu-Shik

2017-04-01

The in vitro generation of cell-based three dimensional (3D) nerve tissue is an attractive subject to improve graft survival and integration into host tissue for neural tissue regeneration or to model biological events in stem cell differentiation. Although 3D organotypic culture strategies are well established for 3D nerve tissue formation of pluripotent stem cells to study underlying biology in nerve development, cell-based nerve tissues have not been developed using human postnatal stem cells with therapeutic potential. Here, we established a culture strategy for the generation of in vitro cell-based 3D nerve tissue from postnatal stem cells from apical papilla (SCAPs) of teeth, which originate from neural crest-derived ectomesenchyme cells. A stem cell population capable of differentiating into neural cell lineages was generated during the ex vivo expansion of SCAPs in the presence of EGF and bFGF, and SCAPs differentiated into neural cells, showing neural cell lineage-related molecular and gene expression profiles, morphological changes and electrophysical property under neural-inductive culture conditions. Moreover, we showed the first evidence that 3D cell-based nerve-like tissue with axons and myelin structures could be generated from SCAPs via 3D organotypic culture using an integrated bioprocess composed of polyethylene glycol (PEG) microwell-mediated cell spheroid formation and subsequent dynamic culture in a high aspect ratio vessel (HARV) bioreactor. In conclusion, the culture strategy in our study provides a novel approach to develop in vitro engineered nerve tissue using SCAPs and a foundation to study biological events in the neural differentiation of postnatal stem cells. Biotechnol. Bioeng. 2017;114: 903-914. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

PubMed Central

Chen, Yi-Dao; Huang, Shiang-Fu; Wang, Hung-Ming

2015-01-01

To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay. PMID:25654105

Automation of 3D cell culture using chemically defined hydrogels.

PubMed

Rimann, Markus; Angres, Brigitte; Patocchi-Tenzer, Isabel; Braum, Susanne; Graf-Hausner, Ursula

2014-04-01

Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo-like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development.

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

PubMed

Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

2017-01-01

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory †

PubMed Central

Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K.; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V. McNeil; Segarra, Verónica A.

2017-01-01

Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented—one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research. PMID:28861134

Cell Culture as an Alternative in Education.

ERIC Educational Resources Information Center

Nardone, Roland M.

1990-01-01

Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

Strategy for selecting disposable bags for cell culture media applications based on a root-cause investigation.

PubMed

Wood, Joseph; Mahajan, Ekta; Shiratori, Masaru

2013-01-01

The use of disposable bags for cell culture media storage has grown significantly in the past decade. Some of the key advantages of using disposable bags relative to non-disposable containers include increased product throughput, decreased cleaning validation costs, reduced risk of cross contamination and lower facility costs. As the scope of use of disposable bags for cell culture applications increases, problematic bags and scenarios should be identified and addressed to continue improving disposables technologies and meet the biotech industry's needs. In this article, we examine a cell culture application wherein media stored in disposable bags is warmed at 37°C before use for cell culture operations. A problematic bag film was identified through a prospective and retrospective cell culture investigation. The investigation provided information on the scope and variation of the issue with respect to different Chinese hamster ovary (CHO) cell lines, cell culture media, and application-specific parameters. It also led to the development of application-specific test methods and enabled a strategy for disposable bag film testing. The strategy was implemented for qualifying an alternative bag film for use in our processes. In this test strategy, multiple lots of 13 bag film types, encompassing eight vendors were evaluated using a three round, cell culture-based test strategy. The test strategy resulted in the determination of four viable bag film options based on the technical data. The results of this evaluation were used to conclude that a volatile or air-quenched compound, likely generated by gamma irradiation of the problematic bag film, negatively impacted cell culture performance. © 2013 American Institute of Chemical Engineers.

Sensing Cell-Culture Assays with Low-Cost Circuitry.

PubMed

Pérez, Pablo; Huertas, Gloria; Maldonado-Jacobi, Andrés; Martín, María; Serrano, Juan A; Olmo, Alberto; Daza, Paula; Yúfera, Alberto

2018-06-11

An alternative approach for cell-culture end-point protocols is proposed herein. This new technique is suitable for real-time remote sensing. It is based on Electrical Cell-substrate Impedance Spectroscopy (ECIS) and employs the Oscillation-Based Test (OBT) method. Simple and straightforward circuit blocks form the basis of the proposed measurement system. Oscillation parameters - frequency and amplitude - constitute the outcome, directly correlated with the culture status. A user can remotely track the evolution of cell cultures in real time over the complete experiment through a web tool continuously displaying the acquired data. Experiments carried out with commercial electrodes and a well-established cell line (AA8) are described, obtaining the cell number in real time from growth assays. The electrodes have been electrically characterized along the design flow in order to predict the system performance and the sensitivity curves. Curves for 1-week cell growth are reported. The obtained experimental results validate the proposed OBT for cell-culture characterization. Furthermore, the proposed electrode model provides a good approximation for the cell number and the time evolution of the studied cultures.

Universal lab-on-a-chip platform for complex, perfused 3D cell cultures

NASA Astrophysics Data System (ADS)

Sonntag, F.; Schmieder, F.; Ströbel, J.; Grünzner, S.; Busek, M.; Günther, K.; Steege, T.; Polk, C.; Klotzbach, U.

2016-03-01

The miniaturization, rapid prototyping and automation of lab-on-a-chip technology play nowadays a very important role. Lab-on-a-chip technology is successfully implemented not only for environmental analysis and medical diagnostics, but also as replacement of animals used for the testing of substances in the pharmaceutical and cosmetics industries. For that purpose the Fraunhofer IWS and partners developed a lab-on-a-chip platform for perfused cell-based assays in the last years, which includes different micropumps, valves, channels, reservoirs and customized cell culture modules. This technology is already implemented for the characterization of different human cell cultures and organoids, like skin, liver, endothelium, hair follicle and nephron. The advanced universal lab-on-a-chip platform for complex, perfused 3D cell cultures is divided into a multilayer basic chip with integrated micropump and application-specific 3D printed cell culture modules. Moreover a technology for surface modification of the printed cell culture modules by laser micro structuring and a complex and flexibly programmable controlling device based on an embedded Linux system was developed. A universal lab-on-a-chip platform with an optional oxygenator and a cell culture module for cubic scaffolds as well as first cell culture experiments within the cell culture device will be presented. The module is designed for direct interaction with robotic dispenser systems. This offers the opportunity to combine direct organ printing of cells and scaffolds with the microfluidic cell culture module. The characterization of the developed system was done by means of Micro-Particle Image Velocimetry (μPIV) and an optical oxygen measuring system.

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

PubMed

de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

2017-05-01

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Hydrogel-based three-dimensional cell culture for organ-on-a-chip applications.

PubMed

Lee, Seung Hwan; Shim, Kyu Young; Kim, Bumsang; Sung, Jong Hwan

2017-05-01

Recent studies have reported that three-dimensionally cultured cells have more physiologically relevant functions than two-dimensionally cultured cells. Cells are three-dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ-on-a-chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation-based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ-on-a-chip. Last, we also discuss the challenges of hydrogel-based three-dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580-589, 2017. © 2017 American Institute of Chemical Engineers.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

PubMed

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

PubMed Central

Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

Comparison of the uptake of methacrylate-based nanoparticles in static and dynamic in vitro systems as well as in vivo.

PubMed

Rinkenauer, Alexandra C; Press, Adrian T; Raasch, Martin; Pietsch, Christian; Schweizer, Simon; Schwörer, Simon; Rudolph, Karl L; Mosig, Alexander; Bauer, Michael; Traeger, Anja; Schubert, Ulrich S

2015-10-28

Polymer-based nanoparticles are promising drug delivery systems allowing the development of new drug and treatment strategies with reduced side effects. However, it remains a challenge to screen for new and effective nanoparticle-based systems in vitro. Important factors influencing the behavior of nanoparticles in vivo cannot be simulated in screening assays in vitro, which still represent the main tools in academic research and pharmaceutical industry. These systems have serious drawbacks in the development of nanoparticle-based drug delivery systems, since they do not consider the highly complex processes influencing nanoparticle clearance, distribution, and uptake in vivo. In particular, the transfer of in vitro nanoparticle performance to in vivo models often fails, demonstrating the urgent need for novel in vitro tools that can imitate aspects of the in vivo situation more accurate. Dynamic cell culture, where cells are cultured and incubated in the presence of shear stress has the potential to bridge this gap by mimicking key-features of organs and vessels. Our approach implements and compares a chip-based dynamic cell culture model to the common static cell culture and mouse model to assess its capability to predict the in vivo success more accurately, by using a well-defined poly((methyl methacrylate)-co-(methacrylic acid)) and poly((methyl methacrylate)-co-(2-dimethylamino ethylmethacrylate)) based nanoparticle library. After characterization in static and dynamic in vitro cell culture we were able to show that physiological conditions such as cell-cell communication of co-cultured endothelial cells and macrophages as well as mechanotransductive signaling through shear stress significantly alter cellular nanoparticle uptake. In addition, it could be demonstrated by using dynamic cell cultures that the in vivo situation is simulated more accurately and thereby can be applied as a novel system to investigate the performance of nanoparticle systems in vivo more reliable. Copyright © 2015. Published by Elsevier B.V.

Preconditioning of mesenchymal stromal cells toward nucleus pulposus-like cells by microcryogels-based 3D cell culture and syringe-based pressure loading system.

PubMed

Zeng, Yang; Feng, Siyu; Liu, Wei; Fu, Qinyouen; Li, Yaqian; Li, Xiaokang; Chen, Chun; Huang, Chenyu; Ge, Zigang; Du, Yanan

2017-04-01

To precondition mesenchymal stromal/stem cells (MSCs) with mechanical stimulation may enhance cell survival and functions following implantation in load bearing environment such as nucleus pulposus (NP) in intervertebral disc (IVD). In this study, preconditioning of MSCs toward NP-like cells was achieved in previously developed poly (ethylene glycol) diacrylate (PEGDA) microcryogels (PMs) within a syringe-based three-dimensional (3D) culture system which provided a facile and cost-effective pressure loading approach. PMs loaded with alginate and MSCs could be incubated in a sealable syringe which could be air-compressed to apply pressure loading through a programmable syringe pump. Expression levels of chondrogenic marker genes SOX9, COL II, and ACAN were significantly upregulated in MSCs when pressure loading of 0.2 MPa or 0.8 MPa was implemented. Expression levels of COL I and COL X were downregulated when pressure loading was applied. In a nude mouse model, MSCs loaded in PMs mechanically stimulated for three days were subcutaneously injected using the same culture syringe. Three weeks postinjection, more proteoglycans (PGs) were deposited and more SOX9 and COL II but less COL I and COL X were stained in 0.2 MPa group. Furthermore, injectable MSCs-loaded PMs were utilized in an ex vivo rabbit IVD organ culture model that demonstrated the leak-proof function and enhanced cell retention of PMs assisted cell delivery to a load bearing environment for potential NP regeneration. This microcryogels-based 3D cell culture and syringe-based pressure loading system represents a novel method for 3D cell culture with mechanical stimulation for better function. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 507-520, 2017. © 2015 Wiley Periodicals, Inc.

Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

PubMed

Park, Ji Eun; Park, Min Hee; Kim, Min Seong; Park, Yeo Reum; Yun, Jung Im; Cheong, Hee Tae; Kim, Minseok; Choi, Jung Hoon; Lee, Eunsong; Lee, Seung Tae

2017-12-01

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research. © 2017 International Federation for Cell Biology.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

PubMed

Cerino, Giulia; Gaudiello, Emanuele; Grussenmeyer, Thomas; Melly, Ludovic; Massai, Diana; Banfi, Andrea; Martin, Ivan; Eckstein, Friedrich; Grapow, Martin; Marsano, Anna

2016-01-01

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment. © 2015 Wiley Periodicals, Inc.

3D Cell Culture in Alginate Hydrogels

PubMed Central

Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

2015-01-01

This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue. PMID:27600217

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

PubMed Central

Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

2018-01-01

Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.

PubMed

Halldorsson, Skarphedinn; Lucumi, Edinson; Gómez-Sjöberg, Rafael; Fleming, Ronan M T

2015-01-15

Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

The Use of Multidimensional Image-Based Analysis to Accurately Monitor Cell Growth in 3D Bioreactor Culture

PubMed Central

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells. PMID:22028809

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture.

PubMed

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.

Paper-based microreactor integrating cell culture and subsequent immunoassay for the investigation of cellular phosphorylation.

PubMed

Lei, Kin Fong; Huang, Chia-Hao

2014-12-24

Investigation of cellular phosphorylation and signaling pathway has recently gained much attention for the study of pathogenesis of cancer. Related conventional bioanalytical operations for this study including cell culture and Western blotting are time-consuming and labor-intensive. In this work, a paper-based microreactor has been developed to integrate cell culture and subsequent immunoassay on a single paper. The paper-based microreactor was a filter paper with an array of circular zones for running multiple cell cultures and subsequent immunoassays. Cancer cells were directly seeded in the circular zones without hydrogel encapsulation and cultured for 1 day. Subsequently, protein expressions including structural, functional, and phosphorylated proteins of the cells could be detected by their specific antibodies, respectively. Study of the activation level of phosphorylated Stat3 of liver cancer cells stimulated by IL-6 cytokine was demonstrated by the paper-based microreactor. This technique can highly reduce tedious bioanalytical operation and sample and reagent consumption. Also, the time required by the entire process can be shortened. This work provides a simple and rapid screening tool for the investigation of cellular phosphorylation and signaling pathway for understanding the pathogenesis of cancer. In addition, the operation of the paper-based microreactor is compatible to the molecular biological training, and therefore, it has the potential to be developed for routine protocol for various research areas in conventional bioanalytical laboratories.

Metabolite profiling of microfluidic cell culture conditions for droplet based screening.

PubMed

Bjork, Sara M; Sjostrom, Staffan L; Andersson-Svahn, Helene; Joensson, Haakan N

2015-07-01

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

Agent-Based Computational Modeling of Cell Culture: Understanding Dosimetry In Vitro as Part of In Vitro to In Vivo Extrapolation

EPA Science Inventory

Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assu...

Chitosan-hyaluronan based 3D co-culture platform for studying the crosstalk of lung cancer cells and mesenchymal stem cells.

PubMed

Han, Hao-Wei; Hsu, Shan-Hui

2016-09-15

The controversial roles of mesenchymal stem cells (MSCs) in lung cancer development are not yet resolved because of the lack of an extracellular environment that mimics the tumor microenvironment. Three-dimensional (3D) culture system is an emerging research tool for biomedical applications such as drug screening. In this study, MSCs and human non-small cell lung carcinoma cells (A549) were co-cultured on a thin biomaterial-based substratum (hyaluronan-grafted chitosan, CS-HA; ∼2μm), and they were self-organized into the 3D tumor co-spheroids with core-shell structure. The gene expression levels of tumorigenicity markers in cancer cells associated with cancer stemness, epithelial-mesenchymal transition (EMT) property, and cell mobility were up-regulated for more than twofold in the MSC-tumor co-spheroids, through the promoted expression of certain tumor enhancers and the direct cell-cell interaction. To verify the different extents of tumorigenicity, A549 cells or those co-cultured with MSCs were transplanted into zebrafish embryos for evaluation in vivo. The tumorigenicity obtained from the zebrafish xenotransplantation model was consistent with that observed in vitro. These evidences suggest that the CS-HA substrate-based 3D co-culture platform for cancer cells and MSCs may be a convenient tool for studying the cell-cell interaction in a tumor-like microenvironment and potentially for cancer drug testing. Mesenchymal stem cells (MSCs) have been found in several types of tumor tissues. However, the controversial roles of MSCs in cancer development are still unsolved. Chitosan and hyaluronan are commonly used materials in the biomedical field. In the current study, we co-cultured lung cancer cells and MSCs on the planar hyaluronan-grafted chitosan (CS-HA) hybrid substrates, and discovered that lung cancer cells and MSCs were rapidly self-assembled into 3D tumor spheroids with core-shell structure on the substrates after only two days in culture. Therefore, CS-HA based 3D co-culture platform can be applied to exploration of the relationship between cancer cells and MSCs and other cancer-related medical applications such as drug screening. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cell growth and migration under octenidine-antiseptic treatment.

PubMed

Jenull, S; Hojdar, K; Laggner, H; Velimirov, B; Zemann, N; Huettinger, M

2015-06-01

The toxicity of octenidine antiseptics in cultured cells contrasts their good tolerability in tissue. This phenomenon prompted us to examine which cell culture conditions allow survival and proliferation and to investigate a possible modulation of toxicity by the extracellular matrix proteoglycan chondroitin sulfate. We tested fibroblasts and MCF7 cells for growth using the MTT test, and assessed wound healing potency with a laceration assay. Expression levels of the genes involved in controlling wound healing were assessed with RT-PCR. A 24 hour exposure to the octenidine-based solution was found incompatible with cell growth. When octenidine solution (0.5-0.5mg/l) was coated on dishes, growth was profoundly reduced after 24 hours, however there was no cytotoxic effect at 0.012 mg/l. Interestingly, when dishes were first coated with chondroitin sulfate the cytotoxicity of octenidine-based solution was modulated. Cell migration was not inhibited by octenidine-based solution treatment (2 minutes; 15 mg/l). No significant changes in gene expression levels in response to the octenidine-based solution treatment were detected. In cell culture conditions application of the octenidine-based solution without toxicity can be observed, comparable to the minimal application required to give full bactericidal effect. Alteration of toxicity by interaction with chondroitin sulfate in cell culture suggests a similar function for extraceullar matrix in intact tissue.

Novel Membrane-Based Electrochemical Sensor for Real-Time Bio-Applications

PubMed Central

Alatraktchi, Fatima AlZahra'a; Bakmand, Tanya; Dimaki, Maria; Svendsen, Winnie E.

2014-01-01

This article presents a novel membrane-based sensor for real-time electrochemical investigations of cellular- or tissue cultures. The membrane sensor enables recording of electrical signals from a cell culture without any signal dilution, thus avoiding loss of sensitivity. Moreover, the porosity of the membrane provides optimal culturing conditions similar to existing culturing techniques allowing more efficient nutrient uptake and molecule release. The patterned sensor electrodes were fabricated on a porous membrane by electron-beam evaporation. The electrochemical performance of the membrane electrodes was characterized by cyclic voltammetry and chronoamperometry, and the detection of synthetic dopamine was demonstrated down to a concentration of 3.1 pM. Furthermore, to present the membrane-sensor functionality the dopamine release from cultured PC12 cells was successfully measured. The PC12 cells culturing experiments showed that the membrane-sensor was suitable as a cell culturing substrate for bio-applications. Real-time measurements of dopamine exocytosis in cell cultures were performed, where the transmitter release was recorded at the point of release. The developed membrane-sensor provides a new functionality to the standard culturing methods, enabling sensitive continuous in vitro monitoring and closely mimicking the in vivo conditions. PMID:25421738

LTCC based bioreactors for cell cultivation

NASA Astrophysics Data System (ADS)

Bartsch, H.; Welker, T.; Welker, K.; Witte, H.; Müller, J.

2016-01-01

LTCC multilayers offer a wide range of structural options and flexibility of connections not available in standard thin film technology. Therefore they are considered as material base for cell culture reactors. The integration of microfluidic handling systems and features for optical and electrical capturing of indicators for cell culture growth offers the platform for an open system concept. The present paper assesses different approaches for the creation of microfluidic channels in LTCC multilayers. Basic functions required for the fluid management in bioreactors include temperature and flow control. Both features can be realized with integrated heaters and temperature sensors in LTCC multilayers. Technological conditions for the integration of such elements into bioreactors are analysed. The temperature regulation for the system makes use of NTC thermistor sensors which serve as real value input for the control of the heater. It allows the adjustment of the fluid temperature with an accuracy of 0.2 K. The tempered fluid flows through the cell culture chamber. Inside of this chamber a thick film electrode array monitors the impedance as an indicator for the growth process of 3-dimensional cell cultures. At the system output a flow sensor is arranged to monitor the continual flow. For this purpose a calorimetric sensor is implemented, and its crucial design parameters are discussed. Thus, the work presented gives an overview on the current status of LTCC based fluid management for cell culture reactors, which provides a promising base for the automation of cell culture processes.

Cell-derived micro-environment helps dental pulp stem cells promote dental pulp regeneration.

PubMed

Zhang, Xuexin; Li, Hui; Sun, Jingjing; Luo, Xiangyou; Yang, Hefeng; Xie, Li; Yang, Bo; Guo, Weihua; Tian, Weidong

2017-10-01

The function of the dental pulp is closely connected to the extracellular matrix (ECM) structure, and ECM has received significant attention due to its biological functions for regulating cells. As such, the interaction between the ECM niche and cells is worth exploring for potential clinical uses. In this study, dental pulp stem cell (DPSC)-derived ECM (DPM) was prepared through cell culture and decellularization to function as the cell niche, and changes in DPSC behaviour and histological analysis of dental pulp tissue regeneration were evaluated following the DPM culture. DPM promoted the replication of DPSCs and exhibited retention of their mineralization. Then, the DPM-based culture strategy under odontogenic culture medium was further investigated, and the mineralization-related markers showed that DPSCs were regulated towards odontogenic differentiation. Dental pulp-like tissue with well-arranged ECM was harvested after a 2-month subcutaneous implantation in nude mice with DPM application. Additionally, DPSCs cultured on the plastic culture surface showed the up-regulation of mineralization makers in vitro, but there was a disorder in matrix formation and mineralization when the cells were cultured in vivo. DPM-based cultivation could serve as a cell niche and modulate DPSC behaviour, and this method also provided an alternative to harvest tissue-specific ECM and provided a strategy for ECM-cell interaction. © 2017 John Wiley & Sons Ltd.

Challenges and opportunities in the manufacture and expansion of cells for therapy.

PubMed

Maartens, Joachim H; De-Juan-Pardo, Elena; Wunner, Felix M; Simula, Antonio; Voelcker, Nicolas H; Barry, Simon C; Hutmacher, Dietmar W

2017-10-01

Laboratory-based ex vivo cell culture methods are largely manual in their manufacturing processes. This makes it extremely difficult to meet regulatory requirements for process validation, quality control and reproducibility. Cell culture concepts with a translational focus need to embrace a more automated approach where cell yields are able to meet the quantitative production demands, the correct cell lineage and phenotype is readily confirmed and reagent usage has been optimized. Areas covered: This article discusses the obstacles inherent in classical laboratory-based methods, their concomitant impact on cost-of-goods and that a technology step change is required to facilitate translation from bed-to-bedside. Expert opinion: While traditional bioreactors have demonstrated limited success where adherent cells are used in combination with microcarriers, further process optimization will be required to find solutions for commercial-scale therapies. New cell culture technologies based on 3D-printed cell culture lattices with favourable surface to volume ratios have the potential to change the paradigm in industry. An integrated Quality-by-Design /System engineering approach will be essential to facilitate the scaled-up translation from proof-of-principle to clinical validation.

SU-8 based microdevices to study self-induced chemotaxis in 3D microenvironments

NASA Astrophysics Data System (ADS)

Ayuso, Jose; Monge, Rosa; Llamazares, Guillermo; Moreno, Marco; Agirregabiria, Maria; Berganzo, Javier; Doblaré, Manuel; Ochoa, Iñaki; Fernandez, Luis

2015-05-01

Tissues are complex three-dimensional structures in which cell behaviour is frequently guided by chemotactic signals. Although starvation and nutrient restriction induce many different chemotactic processes, the recreation of such conditions in vitro remains difficult when using standard cell culture equipment. Recently, microfluidic techniques have arisen as powerful tools to mimic such physiological conditions. In this context, microfluidic three-dimensional cell culture systems require precise control of cell/hydrogel location because samples need to be placed within a microchamber without obstruction of surrounding elements. In this article, SU-8 is studied as structural material for the fabrication of complex cell culture devices due to its good mechanical properties, low gas permeability and sensor integration capacity. In particular, this manuscript presents a SU-8 based microdevice designed to create “self-induced” medium starvation, based on the combination of nutrient restriction and natural cell metabolism. Results show a natural migratory response towards nutrient source, showing how cells adapt to their own microenvironment modifications. The presented results demonstrate the SU-8 potential for microdevice fabrication applied to cell culture.

Co-culture of Adult Mesenchymal Stem Cells and Nucleus Pulposus Cells in Bilaminar Pellets for Intervertebral Disc Regeneration.

PubMed

Allon, Aliza A; Schneider, Richard A; Lotz, Jeffrey C

2009-01-01

Our goal is to optimize stem cell-based tissue engineering strategies in the context of the intervertebral disc environment. We explored the benefits of co-culturing nucleus pulposus cells (NPC) and adult mesenchymal stem cells (MSC) using a novel spherical bilaminar pellet culture system where one cell type is enclosed in a sphere of the other cell type. Our 3D system provides a structure that exploits embryonic processes such as tissue induction and condensation. We observed a unique phenomenon: the budding of co-culture pellets and the formation of satellite pellets that separate from the main pellet. MSC and NPC co-culture pellets were formed with three different structural organizations. The first had random organization. The other two had bilaminar organization with either MSC inside and NPC outside or NPC inside and MSC outside. By 14 days, all co-culture pellets exhibited budding and spontaneously generated satellite pellets. The satellite pellets were composed of both cell types and, surprisingly, all had the same bilaminar organization with MSC on the inside and NPC on the outside. This organization was independent of the structure of the main pellet that the satellites stemmed from. The main pellets generated satellite pellets that spontaneously organized into a bilaminar structure. This implies that structural organization occurs naturally in this cell culture system and may be inherently favorable for cell-based tissue engineering strategies. The occurrence of budding and the organization of satellite pellets may have important implications for the use of co-culture pellets in cell-based therapies for disc regeneration. From a therapeutic point of view, the generation of satellite pellets may be a beneficial feature that would serve to spread donor cells throughout the host matrix and restore normal matrix composition in a sustainable way, ultimately renewing tissue function.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy.

PubMed

Guo, Haiying; Xing, Yizhan; Zhang, Yiming; He, Long; Deng, Fang; Ma, Xiaogen; Li, Yuhong

2018-01-01

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

PubMed Central

Zhang, Yiming; He, Long; Deng, Fang; Ma, Xiaogen

2018-01-01

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells. PMID:29383288

Quantifying oxygen in paper-based cell cultures with luminescent thin film sensors.

PubMed

Boyce, Matthew W; Kenney, Rachael M; Truong, Andrew S; Lockett, Matthew R

2016-04-01

Paper-based scaffolds are an attractive material for generating 3D tissue-like cultures because paper is readily available and does not require specialized equipment to pattern, cut, or use. By controlling the exchange of fresh culture medium with the paper-based scaffolds, we can engineer diffusion-dominated environments similar to those found in spheroids or solid tumors. Oxygen tension directly regulates cellular phenotype and invasiveness through hypoxia-inducible transcription factors and also has chemotactic properties. To date, gradients of oxygen generated in the paper-based cultures have relied on cellular response-based readouts. In this work, we prepared a luminescent thin film capable of quantifying oxygen tensions in apposed cell-containing paper-based scaffolds. The oxygen sensors, which are polystyrene films containing a Pd(II) tetrakis(pentafluorophenyl)porphyrin dye, are photostable, stable in culture conditions, and not cytotoxic. They have a linear response for oxygen tensions ranging from 0 to 160 mmHg O2, and a Stern-Volmer constant (K sv) of 0.239 ± 0.003 mmHg O2 (-1). We used these oxygen-sensing films to measure the spatial and temporal changes in oxygen tension for paper-based cultures containing a breast cancer line that was engineered to constitutively express a fluorescent protein. By acquiring images of the oxygen-sensing film and the fluorescently labeled cells, we were able to approximate the oxygen consumption rates of the cells in our cultures.

A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture

PubMed Central

Freund, Nathaniel W.; Croughan, Matthew S.

2018-01-01

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production—termed Lactate Supplementation and Adaptation (LSA) technology—through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications. PMID:29382079

A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture.

PubMed

Freund, Nathaniel W; Croughan, Matthew S

2018-01-28

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production-termed Lactate Supplementation and Adaptation (LSA) technology-through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications.

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

PubMed

Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

2011-11-15

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

Three-Step Method for Proliferation and Differentiation of Human Embryonic Stem Cell (hESC)-Derived Male Germ Cells

PubMed Central

Lim, Jung Jin; Shim, Myung Sun; Lee, Jeoung Eun; Lee, Dong Ryul

2014-01-01

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells. PMID:24690677

Automated, Miniaturized and Integrated Quality Control-on-Chip (QC-on-a-Chip) for Advanced Cell Therapy Applications

NASA Astrophysics Data System (ADS)

Wartmann, David; Rothbauer, Mario; Kuten, Olga; Barresi, Caterina; Visus, Carmen; Felzmann, Thomas; Ertl, Peter

2015-09-01

The combination of microfabrication-based technologies with cell biology has laid the foundation for the development of advanced in vitro diagnostic systems capable of evaluating cell cultures under defined, reproducible and standardizable measurement conditions. In the present review we describe recent lab-on-a-chip developments for cell analysis and how these methodologies could improve standard quality control in the field of manufacturing cell-based vaccines for clinical purposes. We highlight in particular the regulatory requirements for advanced cell therapy applications using as an example dendritic cell-based cancer vaccines to describe the tangible advantages of microfluidic devices that overcome most of the challenges associated with automation, miniaturization and integration of cell-based assays. As its main advantage lab-on-a-chip technology allows for precise regulation of culturing conditions, while simultaneously monitoring cell relevant parameters using embedded sensory systems. State-of-the-art lab-on-a-chip platforms for in vitro assessment of cell cultures and their potential future applications for cell therapies and cancer immunotherapy are discussed in the present review.

Bioreactor design for successive culture of anchorage-dependent cells operated in an automated manner.

PubMed

Kino-Oka, Masahiro; Ogawa, Natsuki; Umegaki, Ryota; Taya, Masahito

2005-01-01

A novel bioreactor system was designed to perform a series of batchwise cultures of anchorage-dependent cells by means of automated operations of medium change and passage for cell transfer. The experimental data on contamination frequency ensured the biological cleanliness in the bioreactor system, which facilitated the operations in a closed environment, as compared with that in flask culture system with manual handlings. In addition, the tools for growth prediction (based on growth kinetics) and real-time growth monitoring by measurement of medium components (based on small-volume analyzing machinery) were installed into the bioreactor system to schedule the operations of medium change and passage and to confirm that culture proceeds as scheduled, respectively. The successive culture of anchorage-dependent cells was conducted with the bioreactor running in an automated way. The automated bioreactor gave a successful culture performance with fair accordance to preset scheduling based on the information in the latest subculture, realizing 79- fold cell expansion for 169 h. In addition, the correlation factor between experimental data and scheduled values through the bioreactor performance was 0.998. It was concluded that the proposed bioreactor with the integration of the prediction and monitoring tools could offer a feasible system for the manufacturing process of cultured tissue products.

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

PubMed Central

Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

2016-01-01

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

Development of a gastrointestinal tract microscale cell culture analog to predict drug transport

USDA-ARS?s Scientific Manuscript database

Microscale cell culture analogs (uCCAs) are used to study the metabolism and toxicity of a chemical or drug. These in vitro devices are physical replicas of physiologically based pharmacokinetic models that combine microfabrication and cell culture. The goal of this project is to add an independent ...

Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of c-jun and c-fos in Human Tissue Culture Cells

EPA Science Inventory

Aims: To assess virulence of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. Methods and Results: Aeromonas cells were added to Caco-2 cells at approximately a one to one ratio. After 1, 2 and 3 ...

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

PubMed

Tong, Zhixiang; Martyn, Keir; Yang, Andy; Yin, Xiaolei; Mead, Benjamin E; Joshi, Nitin; Sherman, Nicholas E; Langer, Robert S; Karp, Jeffrey M

2018-02-01

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization of Cell Adhesion on Mg Based Implant Materials by Pre-Incubation under Cell Culture Conditions

PubMed Central

Willumeit, Regine; Möhring, Anneke; Feyerabend, Frank

2014-01-01

Magnesium based implants could revolutionize applications where orthopedic implants such as nails, screws or bone plates are used because they are load bearing and degrade over time. This prevents a second surgery to remove conventional implants. To improve the biocompatibility we studied here if and for how long a pre-incubation of the material under cell culture conditions is favorable for cell attachment and proliferation. For two materials, Mg and Mg10Gd1Nd, we could show that 6 h pre-incubation are already enough to form a natural protective layer suitable for cell culture. PMID:24857908

Optimization of cell adhesion on mg based implant materials by pre-incubation under cell culture conditions.

PubMed

Willumeit, Regine; Möhring, Anneke; Feyerabend, Frank

2014-05-05

Magnesium based implants could revolutionize applications where orthopedic implants such as nails, screws or bone plates are used because they are load bearing and degrade over time. This prevents a second surgery to remove conventional implants. To improve the biocompatibility we studied here if and for how long a pre-incubation of the material under cell culture conditions is favorable for cell attachment and proliferation. For two materials, Mg and Mg10Gd1Nd, we could show that 6 h pre-incubation are already enough to form a natural protective layer suitable for cell culture.

Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-09-01

Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture

PubMed Central

Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2017-01-01

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation. PMID:28783133

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture.

PubMed

Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2017-08-07

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase ( p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation.

3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy

NASA Astrophysics Data System (ADS)

Berdeu, Anthony; Laperrousaz, Bastien; Bordy, Thomas; Morales, S.; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric

2018-02-01

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as 5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell 'leaders', migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.

Microfluidic systems for stem cell-based neural tissue engineering.

PubMed

Karimi, Mahdi; Bahrami, Sajad; Mirshekari, Hamed; Basri, Seyed Masoud Moosavi; Nik, Amirala Bakhshian; Aref, Amir R; Akbari, Mohsen; Hamblin, Michael R

2016-07-05

Neural tissue engineering aims at developing novel approaches for the treatment of diseases of the nervous system, by providing a permissive environment for the growth and differentiation of neural cells. Three-dimensional (3D) cell culture systems provide a closer biomimetic environment, and promote better cell differentiation and improved cell function, than could be achieved by conventional two-dimensional (2D) culture systems. With the recent advances in the discovery and introduction of different types of stem cells for tissue engineering, microfluidic platforms have provided an improved microenvironment for the 3D-culture of stem cells. Microfluidic systems can provide more precise control over the spatiotemporal distribution of chemical and physical cues at the cellular level compared to traditional systems. Various microsystems have been designed and fabricated for the purpose of neural tissue engineering. Enhanced neural migration and differentiation, and monitoring of these processes, as well as understanding the behavior of stem cells and their microenvironment have been obtained through application of different microfluidic-based stem cell culture and tissue engineering techniques. As the technology advances it may be possible to construct a "brain-on-a-chip". In this review, we describe the basics of stem cells and tissue engineering as well as microfluidics-based tissue engineering approaches. We review recent testing of various microfluidic approaches for stem cell-based neural tissue engineering.

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

PubMed

Lan, Shih-Feng; Starly, Binil

2011-10-01

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform.

PubMed

Shibuta, Mayu; Tamura, Masato; Kanie, Kei; Yanagisawa, Masumi; Matsui, Hirofumi; Satoh, Taku; Takagi, Toshiyuki; Kanamori, Toshiyuki; Sugiura, Shinji; Kato, Ryuji

2018-06-09

Cellular morphology on and in a scaffold composed of extracellular matrix generally represents the cellular phenotype. Therefore, morphology-based cell separation should be interesting method that is applicable to cell separation without staining surface markers in contrast to conventional cell separation methods (e.g., fluorescence activated cell sorting and magnetic activated cell sorting). In our previous study, we have proposed a cloning technology using a photodegradable gelatin hydrogel to separate the individual cells on and in hydrogels. To further expand the applicability of this photodegradable hydrogel culture platform, we here report an image-based cell separation system imaging cell picker for the morphology-based cell separation on a photodegradable hydrogel. We have developed the platform which enables the automated workflow of image acquisition, image processing and morphology analysis, and collection of a target cells. We have shown the performance of the morphology-based cell separation through the optimization of the critical parameters that determine the system's performance, such as (i) culture conditions, (ii) imaging conditions, and (iii) the image analysis scheme, to actually clone the cells of interest. Furthermore, we demonstrated the morphology-based cloning performance of cancer cells in the mixture of cells by automated hydrogel degradation by light irradiation and pipetting. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system.

PubMed

Mizukami, Amanda; Orellana, Maristela D; Caruso, Sâmia R; de Lima Prata, Karen; Covas, Dimas T; Swiech, Kamilla

2013-01-01

The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10(7) cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10(8) cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical-scale production system. Copyright © 2013 American Institute of Chemical Engineers.

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

PubMed

Birchler, Axel; Berger, Mischa; Jäggin, Verena; Lopes, Telma; Etzrodt, Martin; Misun, Patrick Mark; Pena-Francesch, Maria; Schroeder, Timm; Hierlemann, Andreas; Frey, Olivier

2016-01-19

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

PubMed

Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Density-based clustering analyses to identify heterogeneous cellular sub-populations

NASA Astrophysics Data System (ADS)

Heaster, Tiffany M.; Walsh, Alex J.; Landman, Bennett A.; Skala, Melissa C.

2017-02-01

Autofluorescence microscopy of NAD(P)H and FAD provides functional metabolic measurements at the single-cell level. Here, density-based clustering algorithms were applied to metabolic autofluorescence measurements to identify cell-level heterogeneity in tumor cell cultures. The performance of the density-based clustering algorithm, DENCLUE, was tested in samples with known heterogeneity (co-cultures of breast carcinoma lines). DENCLUE was found to better represent the distribution of cell clusters compared to Gaussian mixture modeling. Overall, DENCLUE is a promising approach to quantify cell-level heterogeneity, and could be used to understand single cell population dynamics in cancer progression and treatment.

A Dual-Mode Bioreactor System for Tissue Engineered Vascular Models.

PubMed

Bono, N; Meghezi, S; Soncini, M; Piola, M; Mantovani, D; Fiore, Gianfranco Beniamino

2017-06-01

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells.

PubMed

Ikeda, Kazuhiro; Nagata, Shogo; Okitsu, Teru; Takeuchi, Shoji

2017-06-06

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

Inhibition of Breast Cancer Progression by Blocking Heterocellular Contact Between Epithelial Cells and Fibroblasts

DTIC Science & Technology

2013-04-01

by employing a microfluidic -based compartmentalized 3D co-culture platform enabling both contact-free and contact-associated co-cultures. 15...SUBJECT TERMS Heterocellular contact between cancer cells and stromal fibroblasts, Microfluidics , 3D 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...and human mammary fibroblasts (HMFs) in breast cancer progression by employing a microfluidic - based compartmentalized 3D co-culture platform

Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing.

PubMed

Yu, Linfen; Chen, Michael C W; Cheung, Karen C

2010-09-21

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Moreover, continuous dynamic perfusion allows the establishment of long term cell culture and subsequent multicellular spheroid formation. A droplet-based microfluidic system was used to form alginate beads with entrapped breast tumor cells. After gelation, the alginate beads were trapped in microsieve structures for cell culture in a continuous perfusion system. The alginate environment permitted cell proliferation and the formation of multicellular spheroids was observed. The dose-dependent response of the tumor spheroids to doxorubicin, and anticancer drug, showed multicellular resistance compared to conventional monolayer culture. The microsieve structures maintain constant location of each bead in the same position throughout the device seeding process, cell proliferation and spheroid formation, treatment with drug, and imaging, permitting temporal and spatial tracking.

A microfluidic platform for 3-dimensional cell culture and cell-based assays.

PubMed

Kim, Minseok S; Yeon, Ju Hun; Park, Je-Kyun

2007-02-01

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.

Biomaterials for 4D stem cell culture

PubMed Central

Hilderbrand, Amber M.; Ovadia, Elisa M.; Rehmann, Matthew S.; Kharkar, Prathamesh M.; Guo, Chen; Kloxin, April M.

2017-01-01

Stem cells reside in complex three-dimensional (3D) environments within the body that change with time, promoting various cellular functions and processes such as migration and differentiation. These complex changes in the surrounding environment dictate cell fate yet, until recently, have been challenging to mimic within cell culture systems. Hydrogel-based biomaterials are well suited to mimic aspects of these in vivo environments, owing to their high water content, soft tissue-like elasticity, and often-tunable biochemical content. Further, hydrogels can be engineered to achieve changes in matrix properties over time to better mimic dynamic native microenvironments for probing and directing stem cell function and fate. This review will focus on techniques to form hydrogel-based biomaterials and modify their properties in time during cell culture using select addition reactions, cleavage reactions, or non-covalent interactions. Recent applications of these techniques for the culture of stem cells in four dimensions (i.e., in three dimensions with changes over time) also will be discussed for studying essential stem cell processes. PMID:28717344

Generation of patterned cell co-cultures in silicone tubing using a microelectrode technique and electrostatic assembly.

PubMed

Kaji, Hirokazu; Sekine, Soichiro; Hashimoto, Masahiko; Kawashima, Takeaki; Nishizawa, Matsuhiko

2007-01-01

We report a method for producing patterned cell adhesion inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.

A multilayer microdevice for cell-based high-throughput drug screening

NASA Astrophysics Data System (ADS)

Liu, Chong; Wang, Lei; Xu, Zheng; Li, Jingmin; Ding, Xiping; Wang, Qi; Chunyu, Li

2012-06-01

A multilayer polydimethylsiloxane microdevice for cell-based high-throughput drug screening is described in this paper. This established microdevice was based on a modularization method and it integrated a drug/medium concentration gradient generator (CGG), pneumatic microvalves and a cell culture microchamber array. The CGG was able to generate five steps of linear concentrations with the same outlet flow rate. The medium/drug flowed through CGG and then into the pear-shaped cell culture microchambers vertically. This vertical perfusion mode was used to reduce the impact of the shear stress on the physiology of cells induced by the fluid flow in the microchambers. Pear-shaped microchambers with two arrays of miropillars at each outlet were adopted in this microdevice, which were beneficial to cell distribution. The chemotherapeutics Cisplatin (DDP)-induced Cisplatin-resistant cell line A549/DDP apoptotic experiments were performed well on this platform. The results showed that this novel microdevice could not only provide well-defined and stable conditions for cell culture, but was also useful for cell-based high-throughput drug screening with less reagents and time consumption.

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.

PubMed

Flaibani, Marina; Luni, Camilla; Sbalchiero, Elisa; Elvassore, Nicola

2009-01-01

It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.

Improved function and growth of pancreatic cells in a three-dimensional bioreactor environment.

PubMed

Samuelson, Lisa; Gerber, David A

2013-01-01

Methods of three-dimensional (3D) cell culture have made significant progress in recent years due to a better understanding of cell to cell interactions and the cell's interface with their surrounding environment. We hypothesized that a microgravity 3D culture system would improve upon the growth and function of a pancreatic progenitor cell population. We developed a rotating wall vessel bioreactor and established a culture system using a pancreatic cell line. Cells in the bioreactors showed robust proliferation, enhanced transcriptional signaling, and improved translation of pancreatic genes compared with two-dimensional static culture. Cells also gained the ability to respond to glucose stimulation, which was not observed in the control cultures. These findings suggest that a 3D microgravity bioreactor environment mimics the niche of the pancreas yielding a cell source with potential for cell-based therapy in the treatment of diabetes.

RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

PubMed

Morabito, Caterina; Steimberg, Nathalie; Mazzoleni, Giovanna; Guarnieri, Simone; Fanò-Illic, Giorgio; Mariggiò, Maria A

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

RCCS Bioreactor-Based Modelled Microgravity Induces Significant Changes on In Vitro 3D Neuroglial Cell Cultures

PubMed Central

Mazzoleni, Giovanna; Fanò-Illic, Giorgio; Mariggiò, Maria A.

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions. PMID:25654124

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

PubMed

Rüger, Beate M; Buchacher, Tanja; Giurea, Alexander; Kubista, Bernd; Fischer, Michael B; Breuss, Johannes M

2018-01-01

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix. Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro , and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model. Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA. Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45 + leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures. Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth.

[Short peptides stimulate skin cell regeneration during ageing].

PubMed

Chalisova, N I; Lin'kova, N S; Zhekalov, A N; Orlova, A O; Ryzhak, G A; Khavinson, V Kh

2014-01-01

The actual goal of gerontocosmetology is deal with the research of new effective and harmless low- molecular substances. The influence of LK and AEDG peptides in concentrations 0.05-2.00 ng/ml on organotypic skin cell cultures proliferation in young and old animals were investigated. Peptides stimulated skin fibroblasts proliferation on 29-45% in skin cell cultures of young and old rats. This effect was observed in smaller concentration diapason and level during skin ageing in old cell cultures as compared to young cell cultures. These data open new approach for creation cosmetology substances in the base of LKand AEDG peptides.

Spontaneous in vitro senescence of glioma cells confirmed by an antibody against IDH1R132H.

PubMed

Stoczynska-Fidelus, Ewelina; Och, Waldemar; Rieske, Piotr; Bienkowski, Michal; Banaszczyk, Mateusz; Winiecka-Klimek, Marta; Zieba, Jolanta; Janik, Karolina; Rosiak, Kamila; Treda, Cezary; Stawski, Robert; Radomiak-Zaluska, Anna; Piaskowski, Sylwester

2014-06-01

We have recently suggested that glioblastoma cells become spontaneously senescent in cell culture conditions. The antibody specific against IDH1(R132H) offers the perfect opportunity to verify this hypothesis. We analyzed the features of senescence in 8 glioma cell cultures showing the IDH1(R132H) mutation based on combination of immunocytochemistry, enzymo-cytochemistry, BrdU incorporation assay and real-time microscopic observation. We report that glioma cells showing the IDH1(R132H) mutation become rapidly and spontaneously senescent in vitro. Senescence was observed in both classical and novel serum-free cell culture conditions. Importantly, the senescent IDH1(R132H)-positive cells showed the expression of stemness marker (SOX2). In vitro senescence appeared to be the main reason of the difficulties in any kind culturing of glioma cells. 3D cell cultures prolonged the survival and in vitro proliferation of neoplastic IDH1(R132H)-positive cells, however, did not enhance the stabilization efficiency. Senescence of glioma cells is spontaneously triggered in vitro, which offers the opportunity of potential new therapeutic strategies based on this phenomenon. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Multizone Paper Platform for 3D Cell Cultures

PubMed Central

Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

2011-01-01

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions.

PubMed

He, Xiao-Tao; Wu, Rui-Xin; Xu, Xin-Yue; Wang, Jia; Yin, Yuan; Chen, Fa-Ming

2018-04-15

Accumulating evidence indicates that the physicochemical properties of biomaterials exert profound influences on stem cell fate decisions. However, matrix-based regulation selected through in vitro analyses based on a given cell population do not genuinely reflect the in vivo conditions, in which multiple cell types are involved and interact dynamically. This study constitutes the first investigation of how macrophages (Mφs) in stiffness-tunable transglutaminase cross-linked gelatin (TG-gel) affect the osteogenesis of bone marrow-derived mesenchymal stem cells (BMMSCs). When a single cell type was cultured, low-stiffness TG-gels promoted BMMSC proliferation, whereas high-stiffness TG-gels supported cell osteogenic differentiation. However, Mφs in high-stiffness TG-gels were more likely to polarize toward the pro-inflammatory M1 phenotype. Using either conditioned medium (CM)-based incubation or Transwell-based co-culture, we found that Mφs encapsulated in the low-stiffness matrix exerted a positive effect on the osteogenesis of co-cultured BMMSCs. Conversely, Mφs in high-stiffness TG-gels negatively affected cell osteogenic differentiation. When both cell types were cultured in the same TG-gel type and placed into the Transwell system, the stiffness-related influences of Mφs on BMMSCs were significantly altered; both the low- and high-stiffness matrix induced similar levels of BMMSC osteogenesis. Although the best material parameter for synergistically affecting Mφs and BMMSCs remains unknown, our data suggest that Mφ involvement in the co-culture system alters previously identified material-related influences on BMMSCs, such as matrix stiffness-related effects, which were identified based on a culture system involving a single cell type. Such Mφ-stem cell interactions should be considered when establishing proper matrix parameter-associated cell regulation in the development of biomimetic biomaterials for regenerative applications. The substrate stiffness of a scaffold plays critical roles in modulating both reparative cells, such as mesenchymal stem cells (MSCs), and immune cells, such as macrophages (Mφs). Although the influences of material stiffness on either Mφs or MSCs, have been extensively described, how the two cell types respond to matrix cues to dynamically affect each other in a three-dimensional (3D) biosystem remains largely unknown. Here, we report our findings that, in a platform wherein Mφs and bone marrow-derived MSCs coexist, matrix stiffness can influence stem cell fate through both direct matrix-associated regulation and indirect Mφ-based modulation. Our data support future studies of the MSC-Mφ-matrix interplay in the 3D context to optimize matrix parameters for the development of the next biomaterial. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

PubMed

Kuo, Ching-Te; Wang, Jong-Yueh; Lin, Yu-Fen; Wo, Andrew M; Chen, Benjamin P C; Lee, Hsinyu

2017-06-29

Biomaterial-based tissue culture platforms have emerged as useful tools to mimic in vivo physiological microenvironments in experimental cell biology and clinical studies. We describe herein a three-dimensional (3D) tissue culture platform using a polydimethylsiloxane (PDMS)-based hanging drop array (PDMS-HDA) methodology. Multicellular spheroids can be achieved within 24 h and further boosted by incorporating collagen fibrils in PDMS-HDA. In addition, the spheroids generated from different human tumor cells exhibited distinct sensitivities toward drug chemotherapeutic agents and radiation as compared with two-dimensional (2D) cultures that often lack in vivo-like biological insights. We also demonstrated that multicellular spheroids may enable key hallmarks of tissue-based bioassays, including drug screening, tumor dissemination, cell co-culture, and tumor invasion. Taken together, these results offer new opportunities not only to achieve the active control of 3D multicellular spheroids on demand, but also to establish a rapid and cost-effective platform to study anti-cancer therapeutics and tumor microenvironments.

Preparation of Cytokine-activated NK Cells for Use in Adoptive Cell Therapy in Cancer Patients: Protocol Optimization and Therapeutic Potential.

PubMed

van Ostaijen-ten Dam, Monique M; Prins, Henk-Jan; Boerman, Gerharda H; Vervat, Carly; Pende, Daniela; Putter, Hein; Lankester, Arjan; van Tol, Maarten J D; Zwaginga, Jaap J; Schilham, Marco W

2016-01-01

Cell-based immunotherapy using donor-derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine-activated NK-cell product. NK cells were isolated either by double depletion (CD3(-), CD19(-)) or by sequential depletion and enrichment (CD3(-,) CD56(+)) via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL)-2 or IL-15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T-cell contamination. After culture with cytokines, the CD3(-)CD56(+) procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3(-)CD19- procedure. CD69, NKp44, and NKG2A expression were higher on CD3(-)CD56(+) products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL-2, T-cell contamination was lower with IL-15. Therefore, further experiments were performed with CD3(-)CD56(+) products cultured with IL-15. Cryopreservation of IL-15-activated NK cells resulted in a loss of cytotoxicity (>92%), whereas thawing of isolated, uncultured NK cells followed by culture with IL-15 yielded cells with about 43% of the original lytic activity. Five-day IL-15-activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell–based immunotherapeutic strategies in a clinical setting.

Sensor Access to the Cellular Microenvironment Using the Sensing Cell Culture Flask.

PubMed

Kieninger, Jochen; Tamari, Yaara; Enderle, Barbara; Jobst, Gerhard; Sandvik, Joe A; Pettersen, Erik O; Urban, Gerald A

2018-04-26

The Sensing Cell Culture Flask (SCCF) is a cell culture monitoring system accessing the cellular microenvironment in 2D cell culture using electrochemical microsensors. The system is based on microfabricated sensor chips embedded in standard cell culture flasks. Ideally, the sensor chips could be equipped with any electrochemical sensor. Its transparency allows optical inspection of the cells during measurement. The surface of the sensor chip is in-plane with the flask surface allowing undisturbed cell growth on the sensor chip. A custom developed rack system allows easy usage of multiple flasks in parallel within an incubator. The presented data demonstrates the application of the SCCF with brain tumor (T98G) and breast cancer (T-47D) cells. Amperometric oxygen sensors were used to monitor cellular respiration with different incubation conditions. Cellular acidification was accessed with potentiometric pH sensors using electrodeposited iridium oxide films. The system itself provides the foundation for electrochemical monitoring systems in 3D cell culture.

Polysaccharide-based hydrogels with tunable composition as 3D cell culture systems.

PubMed

Gentilini, Roberta; Munarin, Fabiola; Bloise, Nora; Secchi, Eleonora; Visai, Livia; Tanzi, Maria Cristina; Petrini, Paola

2018-04-01

To date, cell cultures have been created either on 2-dimensional (2D) polystyrene surfaces or in 3-dimensional (3D) systems, which do not offer a controlled chemical composition, and which lack the soft environment encountered in vivo and the chemical stimuli that promote cell proliferation and allow complex cellular behavior. In this study, pectin-based hydrogels were developed and are proposed as versatile cell culture systems. Pectin-based hydrogels were produced by internally crosslinking pectin with calcium carbonate at different initial pH, aiming to control crosslinking kinetics and degree. Additionally, glucose and glutamine were added as additives, and their effects on the viscoelastic properties of the hydrogels and on cell viability were investigated. Pectin hydrogels showed in high cell viability and shear-thinning behavior. Independently of hydrogel composition, an initial swelling was observed, followed by a low percentage of weight variation and a steady-state stage. The addition of glucose and glutamine to pectin-based hydrogels rendered higher cell viability up to 90%-98% after 1 hour of incubation, and these hydrogels were maintained for up to 7 days of culture, yet no effect on viscoelastic properties was detected. Pectin-based hydrogels that offer tunable composition were developed successfully. They are envisioned as synthetic extracellular matrix (ECM) either to study complex cellular behaviors or to be applied as tissue engineering substitutes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygenmore » and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high-content screen for the identification of compounds that target cells in dormant tumor spheroid regions. • Metabolite profiling by NMR spectroscopy on 3D tumor spheroids. • Identification of respiratory chain inhibitors to specifically induce cell death in inner tumor spheroid regions. • Respiratory chain inhibitors enhance cytostatic based therapy in vitro.« less

Production of oncolytic adenovirus and human mesenchymal stem cells in a single-use, Vertical-Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier-based cell culture processes.

PubMed

Sousa, Marcos F Q; Silva, Marta M; Giroux, Daniel; Hashimura, Yas; Wesselschmidt, Robin; Lee, Brian; Roldão, António; Carrondo, Manuel J T; Alves, Paula M; Serra, Margarida

2015-01-01

Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers.

Recent developments in processing systems for cell and tissue cultures toward therapeutic application.

PubMed

Kino-oka, Masahiro; Taya, Masahito

2009-10-01

Innovative techniques of cell and tissue processing, based on tissue engineering, have been developed for therapeutic applications. Cell expansion and tissue reconstruction through ex vivo cultures are core processes used to produce engineered tissues with sufficient structural integrity and functionality. In manufacturing, strict management against contamination and human error is compelled due to direct use of un-sterilable products and the laboriousness of culture operations, respectively. Therefore, the development of processing systems for cell and tissue cultures is one of the critical issues for ensuring a stable process and quality of therapeutic products. However, the siting criterion of culture systems to date has not been made clear. This review article classifies some of the known processing systems into 'sealed-chamber' and 'sealed-vessel' culture systems based on the difference in their aseptic spaces, and describes the potential advantages of these systems and current states of culture systems, especially those established by Japanese companies. Moreover, on the basis of the guidelines for isolator systems used in aseptic processing for healthcare products, which are issued by the International Organization for Standardization, the siting criterion of the processing systems for cells and tissue cultures is discussed in perspective of manufacturing therapeutic products in consideration of the regulations according to the Good Manufacturing Practice.

Soft material-based microculture system having air permeable cover sheet for the protoplast culture of Nicotiana tabacum.

PubMed

Ju, Jong Il; Ko, Jung-Moon; Kim, So Hyeon; Baek, Ju Yeoul; Cha, Hyeon-Cheol; Lee, Sang Hoon

2006-08-01

In plant cell culture, the delivery of nutrition and gas (mainly oxygen) to the cells is the most important factor for viability. In this paper, we propose a polydimethylsiloxane (PDMS)-based microculture system that is designed to have good aeration. PDMS is known to have excellent air permeability, and through the experimental method, we investigated the relation between the degree of air delivery and the thickness of the PDMS sheet covering the culture chamber. We determined the proper thickness of the cover sheet, and cultured protoplasts of Nicotiana tabacum in a culture chamber covered with a PDMS sheet having thickness of 400 microm. The cells were successfully divided, and lived well inside the culture chamber for 10 days. In addition, protoplasts were cultured inside the culture chambers covered with the cover glass and the PDMS sheet, respectively, and the microcolonies were formed well inside the PDMS covered chamber after 10 days.

Dispersible oxygen microsensors map oxygen gradients in three-dimensional cell cultures.

PubMed

Lesher-Pérez, Sasha Cai; Kim, Ge-Ah; Kuo, Chuan-Hsien; Leung, Brendan M; Mong, Sanda; Kojima, Taisuke; Moraes, Christopher; Thouless, M D; Luker, Gary D; Takayama, Shuichi

2017-09-26

Phase fluorimetry, unlike the more commonly used intensity-based measurement, is not affected by differences in light paths from culture vessels or by optical attenuation through dense 3D cell cultures and hydrogels thereby minimizing dependence on signal intensity for accurate measurements. This work describes the use of phase fluorimetry on oxygen-sensor microbeads to perform oxygen measurements in different microtissue culture environments. In one example, cell spheroids were observed to deplete oxygen from the cell-culture medium filling the bottom of conventional microwells within minutes, whereas oxygen concentrations remained close to ambient levels for several days in hanging-drop cultures. By dispersing multiple oxygen microsensors in cell-laden hydrogels, we also mapped cell-generated oxygen gradients. The spatial oxygen mapping was sufficiently precise to enable the use of computational models of oxygen diffusion and uptake to give estimates of the cellular oxygen uptake rate and the half-saturation constant. The results show the importance of integrated design and analysis of 3D cell cultures from both biomaterial and oxygen supply aspects. While this paper specifically tests spheroids and cell-laden gel cultures, the described methods should be useful for measuring pericellular oxygen concentrations in a variety of biomaterials and culture formats.

Phenotypic feature quantification of patient derived 3D cancer spheroids in fluorescence microscopy image

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Rhee, Seon-Min; Seo, Ji-Hyun; Kim, Myoung-Hee

2017-03-01

Patients' responses to a drug differ at the cellular level. Here, we present an image-based cell phenotypic feature quantification method for predicting the responses of patient-derived glioblastoma cells to a particular drug. We used high-content imaging to understand the features of patient-derived cancer cells. A 3D spheroid culture formation resembles the in vivo environment more closely than 2D adherent cultures do, and it allows for the observation of cellular aggregate characteristics. However, cell analysis at the individual level is more challenging. In this paper, we demonstrate image-based phenotypic screening of the nuclei of patient-derived cancer cells. We first stitched the images of each well of the 384-well plate with the same state. We then used intensity information to detect the colonies. The nuclear intensity and morphological characteristics were used for the segmentation of individual nuclei. Next, we calculated the position of each nucleus that is appeal of the spatial pattern of cells in the well environment. Finally, we compared the results obtained using 3D spheroid culture cells with those obtained using 2D adherent culture cells from the same patient being treated with the same drugs. This technique could be applied for image-based phenotypic screening of cells to determine the patient's response to the drug.

Proteomic comparison of 3D and 2D glioma models reveals increased HLA-E expression in 3D models is associated with resistance to NK cell-mediated cytotoxicity.

PubMed

He, Weiqi; Kuang, Yongqin; Xing, Xuemin; Simpson, Richard J; Huang, Haidong; Yang, Tao; Chen, Jingmin; Yang, Libin; Liu, Enyu; He, Weifeng; Gu, Jianwen

2014-05-02

Three-dimensional cell culture techniques can better reflect the in vivo characteristics of tumor cells compared with traditional monolayer cultures. Compared with their 2D counterparts, 3D-cultured tumor cells showed enhanced resistance to the cytotoxic T cell-mediated immune response. However, it remains unclear whether 3D-cultured tumor cells have an enhanced resistance to NK cell cytotoxicity. In this study, a total of 363 differentially expressed proteins were identified between the 2D- and 3D-cultured U251 cells by comparative proteomics, and an immune-associated protein-protein interaction (PPI) network based on these differential proteins was constructed by bioinformatics. Within the network, HLA-E, as a molecule for inhibiting NK cell activation, was significantly up-regulated in the 3D-cultured tumor cells. Then, we found that the 3D-cultured U251 cells exhibited potent resistance to NK cell cytotoxicity in vitro and were prone to tumor formation in vivo. The resistance of the 3D-cultured tumor cells to NK cell lysis was mediated by the HLA-E/NKG2A interaction because the administration of antibodies that block either HLA-E or NKG2A completely eliminated this resistance and significantly decreased tumor formation. Taken together, our findings indicate that HLA-E up-regulation in 3D-cultured cells may result in enhanced tumor resistance to NK cell-mediated immune response.

Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds

NASA Technical Reports Server (NTRS)

Goldstein, A. S.; Juarez, T. M.; Helmke, C. D.; Gustin, M. C.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

2001-01-01

Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.

Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

PubMed

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.

PubMed

Dainiak, Maria B; Savina, Irina N; Musolino, Isabella; Kumar, Ashok; Mattiasson, Bo; Galaev, Igor Yu

2008-01-01

Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests.

Human neuroblastoma (SH-SY5Y) cell culture and differentiation in 3-D collagen hydrogels for cell-based biosensing.

PubMed

Desai, Anu; Kisaalita, William S; Keith, Charles; Wu, Z-Z

2006-02-15

Cell-based three-dimensional systems are desirable in the field of high throughput screening assays due to their potential similarity to in vivo environment. We have used SH-SY5Y human neuroblastoma cells cultured in 3-D collagen hydrogel, confocal microscopy and immunofluorescence staining, to assess the merit of the system as a functional, cell-based biosensor. Our results show differences between 2-D and 3-D resting membrane potential development profile upon differentiation. There was no statistically significant difference in SH-SY5Y proliferation rate between 2-D monolayer and 3-D collagen culture formats. A large percentage of cells (2-D, 91.30% and 3-D, 84.93%) did not develop resting membrane potential value equal to or lower than -40 mV; instead cells exhibited a heterogeneous resting membrane potential distribution. In response to high K(+) (50 mM) depolarization, 3-D cells were less responsive in terms of increase in intracellular Ca(2+), in comparison to 2-D cells, supporting the hypothesis that 2-D cell calcium dynamics may be exaggerated. L-Type Ca(2+) expression levels based on staining results was inconsistent with Bay K 8644 channel activation results, strongly suggesting that either the majority of the channels were non-functional or could not be activated by Bay K 8644. In general, the results in this study confirm the depolarization-induced differences in intracellular calcium release when cultured using a 2-D versus a 3-D matrix.

Extraction and Estimation of Secondary Metabolites from Date Palm Cell Suspension Cultures.

PubMed

Naik, Poornananda M; Al-Khayri, Jameel M

2017-01-01

The health benefits of dates arise from their content of phytochemicals, known for having pharmacological properties, including flavonoids, carotenoids, phenolic acids, sterols, procyanidins, and anthocyanins. In vitro cell culture technology has become an attractive means for the production of biomass and bioactive compounds. This chapter describes step-by-step procedures for the induction and proliferation of callus from date palm offshoots on Murashige and Skoog (MS) medium supplemented with plant growth regulators. Subsequently cell suspension cultures are established for optimum biomass accumulation, based on the growth curve developed by packed cell volume as well as fresh and dry weights. The highest production of biomass occurs at the 11th week after culturing. Moreover, this chapter describes methodologies for the extraction and analysis of secondary metabolites of date palm cell suspension cultures using high-performance liquid chromatography (HPLC). The optimum level of catechin, caffeic acid, apigenin, and kaempferol from the cell suspension cultures establishes after the 11th and 12th weeks of culture. This protocol is useful for scale-up production of secondary metabolites from date palm cell suspension cultures.

Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

PubMed Central

2012-01-01

Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material. PMID:22429795

Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production.

PubMed

Montomoli, Emanuele; Khadang, Baharak; Piccirella, Simona; Trombetta, Claudia; Mennitto, Elisa; Manini, Ilaria; Stanzani, Valerio; Lapini, Giulia

2012-05-01

In the 20th century, three influenza pandemics killed approximately 100 million people. The traditional method of influenza vaccine manufacturing is based on using chicken eggs. However, the necessity of the availability of millions of fertile eggs in the event of a pandemic has led research to focus on the development of cell culture-derived vaccines, which offer shorter lead-in times and greater flexibility of production. So far, the cell substrates being evaluated and in use include Vero, Madin-Darby canine kidney, PER.C6 and insect cells. However, Vero cells are the most widely accepted among others. This review introduces briefly the concepts of advanced cell culture-derived influenza vaccine production and highlights the advantages of these vaccines in terms of efficiency, speed and immunogenicity based on the clinical data obtained from different studies.

Immunogenicity is preferentially induced in sparse dendritic cell cultures.

PubMed

Nasi, Aikaterini; Bollampalli, Vishnu Priya; Sun, Meng; Chen, Yang; Amu, Sylvie; Nylén, Susanne; Eidsmo, Liv; Rothfuchs, Antonio Gigliotti; Réthi, Bence

2017-03-09

We have previously shown that human monocyte-derived dendritic cells (DCs) acquired different characteristics in dense or sparse cell cultures. Sparsity promoted the development of IL-12 producing migratory DCs, whereas dense cultures increased IL-10 production. Here we analysed whether the density-dependent endogenous breaks could modulate DC-based vaccines. Using murine bone marrow-derived DC models we show that sparse cultures were essential to achieve several key functions required for immunogenic DC vaccines, including mobility to draining lymph nodes, recruitment and massive proliferation of antigen-specific CD4+ T cells, in addition to their TH1 polarization. Transcription analyses confirmed higher commitment in sparse cultures towards T cell activation, whereas DCs obtained from dense cultures up-regulated immunosuppressive pathway components and genes suggesting higher differentiation plasticity towards osteoclasts. Interestingly, we detected a striking up-regulation of fatty acid and cholesterol biosynthesis pathways in sparse cultures, suggesting an important link between DC immunogenicity and lipid homeostasis regulation.

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

PubMed

Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

2017-03-01

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Counting cell number in situ by quantification of dimethyl sulphide in culture headspace.

PubMed

Chippendale, Thomas W E; Španěl, Patrik; Smith, David; El Haj, Alicia J

2014-10-07

A novel, non-invasive technique is reported for determining the numbers of cells in a culture by quantifying dimethyl sulphide (DMS) in the culture headspace as produced by the cellular enzymatic reduction of dissolved dimethyl sulphoxide (DMSO). Measured DMS concentrations, as performed using selected ion flow tube mass spectrometry (SIFT-MS), in the headspace of 2D and 3D cultures of four cell lines, viz. HEK293 (kidney), MG63 (bone), hepG2 (liver) and CALU-1 (lung), linearly correlate with starting cell number. Clear differences in the rates of production of DMS by the four cell types in both the 2D and 3D situations are seen. This novel analytical technique for cell enumeration offers a significant contribution to quality assessment across cell-based research and industry, including analysis of large scale culture systems, and for routine cell biology research.

Role of heterogeneous cell population on modulation of dendritic cell phenotype and activation of CD8 T cells for use in cell-based immunotherapies.

PubMed

Frizzell, Hannah; Park, Jaehyung; Comandante Lou, Natacha; Woodrow, Kim A

2017-01-01

Dendritic cell (DC)-based immunotherapies have much utility in their ability to prime antigen-specific adaptive immune responses. However, there does not yet exist a consensus standard to how DCs should be primed. In this study, we aimed to determine the role of heterogeneous co-cultures, composed of both CD11c+ (DCs) and CD11c- cells, in combination with monophosphoryl lipid A (MPLA) stimulation on DC phenotype and function. Upon DC priming in different co-culture ratios, we observed reduced expression of MHCII and CD86 and increased antigen uptake among CD11c+ cells in a CD11c- dependent manner. DCs from all culture conditions were induced to mature by MPLA treatment, as determined by secretion of pro-inflammatory cytokines IL-12 and TNF-α. Antigen-specific stimulation of CD4+ T cells was not modulated by co-culture composition, in terms of proliferation nor levels of IFN-γ. However, the presence of CD11c- cells enhanced cross-presentation to CD8+ T cells compared to purified CD11c+ cells, resulting in increased cell proliferation along with higher IFN-γ production. These findings demonstrate the impact of cell populations present during DC priming, and point to the use of heterogeneous cultures of DCs and innate immune cells to enhance cell-mediated immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell culture-based biosensing techniques for detecting toxicity in water.

PubMed

Tan, Lu; Schirmer, Kristin

2017-06-01

The significant increase of contaminants entering fresh water bodies calls for the development of rapid and reliable methods to monitor the aquatic environment and to detect water toxicity. Cell culture-based biosensing techniques utilise the overall cytotoxic response to external stimuli, mediated by a transduced signal, to specify the toxicity of aqueous samples. These biosensing techniques can effectively indicate water toxicity for human safety and aquatic organism health. In this review we account for the recent developments of the mainstream cell culture-based biosensing techniques for water quality evaluation, discuss their key features, potentials and limitations, and outline the future prospects of their development. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Large-scale progenitor cell expansion for multiple donors in a monitored hollow fibre bioreactor.

PubMed

Lambrechts, Toon; Papantoniou, Ioannis; Rice, Brent; Schrooten, Jan; Luyten, Frank P; Aerts, Jean-Marie

2016-09-01

With the increasing scale in stem cell production, a robust and controlled cell expansion process becomes essential for the clinical application of cell-based therapies. The objective of this work was the assessment of a hollow fiber bioreactor (Quantum Cell Expansion System from Terumo BCT) as a cell production unit for the clinical-scale production of human periosteum derived stem cells (hPDCs). We aimed to demonstrate comparability of bioreactor production to standard culture flask production based on a product characterization in line with the International Society of Cell Therapy in vitro benchmarks and supplemented with a compelling quantitative in vivo bone-forming potency assay. Multiple process read-outs were implemented to track process performance and deal with donor-to-donor-related variation in nutrient needs and harvest timing. The data show that the hollow fiber bioreactor is capable of robustly expanding autologous hPDCs on a clinical scale (yield between 316 million and 444 million cells starting from 20 million after ± 8 days of culture) while maintaining their in vitro quality attributes compared with the standard flask-based culture. The in vivo bone-forming assay on average resulted in 10.3 ± 3.7% and 11.0 ± 3.8% newly formed bone for the bioreactor and standard culture flask respectively. The analysis showed that the Quantum system provides a reproducible cell expansion process in terms of yields and culture conditions for multiple donors. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

"Artificial micro organs"--a microfluidic device for dielectrophoretic assembly of liver sinusoids.

PubMed

Schütte, Julia; Hagmeyer, Britta; Holzner, Felix; Kubon, Massimo; Werner, Simon; Freudigmann, Christian; Benz, Karin; Böttger, Jan; Gebhardt, Rolf; Becker, Holger; Stelzle, Martin

2011-06-01

In order to study possible toxic side effects of potential drug compounds in vitro a reliable test system is needed. Predicting liver toxicity presents a major challenge of particular importance as liver cells grown in a cell culture suffer from a rapid loss of their liver specific functions. Therefore we are developing a new microfluidic test system for liver toxicity. This test system is based on an organ-like liver 3D co-culture of hepatocytes and endothelial cells. We devised a microfluidic chip featuring cell culture chambers with integrated electrodes for the assembly of liver sinusoids by dielectrophoresis. Fluid channels enable an organ-like perfusion with culture media and test compounds. Different chamber designs were studied and optimized with regard to dielectrophoretic force distribution, hydrodynamic flow profile, and cell trapping rate using numeric simulations. Based on simulation results a microchip was injection-moulded from COP. This chip allowed the assembly of viable hepatocytes and endothelial cells in a sinusoid-like fashion.

Culture of human anulus fibrosus cells on polyamide nanofibers: extracellular matrix production.

PubMed

Gruber, Helen E; Hoelscher, Gretchen; Ingram, Jane A; Hanley, Edward N

2009-01-01

Studies were approved by the authors' Human Subjects Institutional Review Board. Human anulus cells were tested for growth and extracellular matrix (ECM) production in vitro. To investigate cell attachment, cell proliferation, and ECM production of human intervertebral disc anulus cells seeded onto randomly oriented electrospun polyamide nanofibers. Because nanofibrillar matrices have the potential to promote microenvironments, which may mimic in vivo conditions and resemble connective tissue, their utilization opens new avenues for cell-based tissue engineering applications for disc cells. Anulus cells were isolated from 4 cervical spine surgical disc specimens, expanded, and seeded into either routine plastic culture (control) or a nanofiber surface of randomly oriented electrospun polyamide nanofibers (Ultra-Web-coated culture dish, Corning) with a positive charge or without a charge. Cells were cultured for 9 days, digital images captured, cells harvested, embedded in paraffin, and examined for production of extracellular matrix (ECM). Additional anulus cultures were tested to quantitatively assess total proteoglycan production and cell proliferation under control or nanofiber cultures. Cells attached well and exhibited cell extensions within the nanofiber layers; cells on the charged nanofiber surface deposited greater amounts of chondroitin sulfate than of type II collagen than cells cultured on the uncharged nanofiber surface. Results showed that culture of anulus cells on nanofibers was permissive for secretion and assembly of type II collagen and chondroitin sulfate. Significantly greater total proteoglycan formation was present after culture on the nanofiber with added charge conditions {control, 0.6116 microg/mL +/- 0.186 [4] [mean +/- sem(n)] vs. 1.201 +/- 0.2509 [4], P < 0.05}. Cell proliferation, however, did not differ among treatment groups. Culture of anulus cells on nanofibers was found to be permissive for secretion and assembly of type II collagen and chondroitin sulfate, and culture on nanofibers with added charge significantly increased total proteoglycan production. These novel findings point to the need for further examination of nanofibrillar 3D culture of anulus cells for tissue engineering applications.

Multiwell cell culture plate format with integrated microfluidic perfusion system

NASA Astrophysics Data System (ADS)

Domansky, Karel; Inman, Walker; Serdy, Jim; Griffith, Linda G.

2006-01-01

A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

PNIPAAm-based biohybrid injectable hydrogel for cardiac tissue engineering.

PubMed

Navaei, Ali; Truong, Danh; Heffernan, John; Cutts, Josh; Brafman, David; Sirianni, Rachael W; Vernon, Brent; Nikkhah, Mehdi

2016-03-01

Injectable biomaterials offer a non-invasive approach to deliver cells into the myocardial infarct region to maintain a high level of cell retention and viability and initiate the regeneration process. However, previously developed injectable matrices often suffer from low bioactivity or poor mechanical properties. To address this need, we introduced a biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with excellent bioactivity as well as mechanical robustness for cardiac tissue engineering. A unique feature of our work was that we performed extensive in vitro biological analyses to assess the functionalities of cardiomyocytes (CMs) alone and in co-culture with cardiac fibroblasts (CFs) (2:1 ratio) within the hydrogel matrix. The synthesized hydrogel exhibited viscoelastic behavior (storage modulus: 1260 Pa) and necessary water content (75%) to properly accommodate the cardiac cells. The encapsulated cells demonstrated a high level of cell survival (90% for co-culture condition, day 7) and spreading throughout the hydrogel matrix in both culture conditions. A dense network of stained F-actin fibers (∼ 6 × 10(4) μm(2) area coverage, co-culture condition) illustrated the formation of an intact and three dimensional (3D) cell-embedded matrix. Furthermore, immunostaining and gene expression analyses revealed mature phenotypic characteristics of cardiac cells. Notably, the co-culture group exhibited superior structural organization and cell-cell coupling, as well as beating behavior (average ∼ 45 beats per min, co-culture condition, day 7). The outcome of this study is envisioned to open a new avenue for extensive in vitro characterization of injectable matrices embedded with 3D mono- and co-culture of cardiac cells prior to in vivo experiments. In this work, we synthesized a new class of biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with suitable bioactivity and mechanical properties for cardiac tissue engineering. A significant aspect of our work was that we performed extensive in vitro biological analyses to assess the functionality of cardiomyocytes alone and in co-culture with cardiac fibroblasts encapsulated within the 3D hydrogel matrix. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The current status of alternatives to animal testing and predictive toxicology methods using liver microfluidic biochips.

PubMed

Prot, Jean Matthieu; Leclerc, Eric

2012-06-01

In this paper, we will consider new in vitro cell culture platforms and the progress made, based on the microfluidic liver biochips dedicated to pharmacological and toxicological studies. Particular emphasis will be given to recent developments in the microfluidic tools dedicated to cell culture (more particularly liver cell culture), in silico opportunities for Physiologically Based PharmacoKinetic (PBPK) modelling, the challenge of the mechanistic interpretations offered by the approaches resulting from "multi-omics" data (transcriptomics, proteomics, metabolomics, cytomics) and imaging microfluidic platforms. Finally, we will discuss the critical features regarding microfabrication, design and materials, and cell functionality as the key points for the future development of new microfluidic liver biochips.

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

PubMed

Du, Xiaohui; Li, Wanming; Du, Guansheng; Cho, Hansang; Yu, Min; Fang, Qun; Lee, Luke P; Fang, Jin

2018-03-06

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

PubMed

Chen, D; Qu, Y; Hua, X; Zhang, L; Liu, Z; Pflugfelder, S C; Li, D-Q

2017-06-01

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

NASA Technical Reports Server (NTRS)

Schatten, H.; Hedrick, J.; Chakrabarti, A.

1998-01-01

Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

NASA Astrophysics Data System (ADS)

Zhang, Yuanxing

Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

Three-dimensional prostate tumor model based on a hyaluronic acid-alginate hydrogel for evaluation of anti-cancer drug efficacy.

PubMed

Tang, Yadong; Huang, Boxin; Dong, Yuqin; Wang, Wenlong; Zheng, Xi; Zhou, Wei; Zhang, Kun; Du, Zhiyun

2017-10-01

In vitro cell-based assays are widely applied to evaluate anti-cancer drug efficacy. However, the conventional approaches are mostly based on two-dimensional (2D) culture systems, making it difficult to recapitulate the in vivo tumor scenario because of spatial limitations. Here, we develop an in vitro three-dimensional (3D) prostate tumor model based on a hyaluronic acid (HA)-alginate hybrid hydrogel to bridge the gap between in vitro and in vivo anticancer drug evaluations. In situ encapsulation of PCa cells was achieved by mixing HA and alginate aqueous solutions in the presence of cells and then crosslinking with calcium ions. Unlike in 2D culture, cells were found to aggregate into spheroids in a 3D matrix. The expression of epithelial to mesenchyme transition (EMT) biomarkers was found to be largely enhanced, indicating an increased invasion and metastasis potential in the hydrogel matrix. A significant up-regulation of proangiogenic growth factors (IL-8, VEGF) and matrix metalloproteinases (MMPs) was observed in 3D-cultured PCa cells. The results of anti-cancer drug evaluation suggested a higher drug tolerance within the 3D tumor model compared to conventional 2D-cultured cells. Finally, we found that the drug effect within the in vitro 3D cancer model based on HA-alginate matrix exhibited better predictability for in vivo drug efficacy.

Image-based evaluations of distribution and cytotoxicity of Irinotecan (CPT-11) in a multi-compartment micro-cell coculture device.

PubMed

Nakayama, Hidenari; Kimura, Hiroshi; Fujii, Teruo; Sakai, Yasuyuki

2014-06-01

We recently developed a polydimethylsiloxane (PDMS)-based three-compartment microfluidic cocultivation device enabling real-time interactions of different cell populations as an advanced physiologically-relevant cell-based assay. This device had valves and small magnetic stirrer-based internal pumps for easy and flexible perfusion operations. In this study, we applied this device for the evaluation of Irinotecan (CPT-11) toxicity to the lung, because it is detoxified by the liver and accumulated in the fat in humans. We successfully cultured representative three different tissue model cells in each compartment under the individual culture conditions and also in entire perfusion. Growth inhibition of rat lung epithelial cell line L-2, was measured when administered with 50 μM CPT-11 under various cocultivation conditions with respect to the presences and absence of primary rat hepatocytes (liver tissue model) and adipocyte-like cells (fat tissue model) induced from a mouse fibroblast cell line, 3T3-L1. Although CPT-11 showed moderate toxicity to the pure culture of L-2 cells in the device after 72 h of perfusion culture, this was lowered mainly in the presence of the liver tissue. Inhibition of the L-2 cell growth agreed with the area under curve (AUC) values obtained from fluorescent image-based analyses in each compartment. These results demonstrate that developed simple and flexible microfluidic cocultivation device, with appropriate image-based analyses, can be used in evaluating toxicokinetic behaviors of drug candidates in systemic levels. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Continuous Non-Destructive Monitoring of Cell Health Using Impedance Based Interdigitated Electrode Structured Sensors

NASA Astrophysics Data System (ADS)

Paschero, Anna; McLoughlin, Eve; Moore, Eric

2011-06-01

This article examines some preliminary tests which were performed in order to evaluate the best electrode configuration (width and spacing) for cell culture analyses. Biochips packaged with indium tin oxide (ITO) interdigitated electrodes (IDEs) were used to perform impedance measurements on A549 cells cultured on the surface of the biochip. Several tests were carried out using a 10 mM solution of Sodium Chloride (NaCl), cell medium and the cell culture itself to characterize some of the configurations already fabricated in the facilities at Tyndall National Institute.

Cell growth and protein expression of Shewanella oneidensis in biofilms and hydrogel-entrapped cultures.

PubMed

Zhang, Yingdan; Ng, Chun Kiat; Cohen, Yehuda; Cao, Bin

2014-05-01

The performance of biofilm-based bioprocesses is difficult to predict and control because of the intrinsic heterogeneous and dynamic properties of microbial biofilms. Biofilm mimics, such as microbial cells entrapped in polymeric scaffolds that are permeable for nutrients, have been proposed to replace real biofilms to achieve long-term robust performance in engineering applications. However, the physiological differences between cells that are physically entrapped in a synthetic polymeric matrix and biofilm cells that are encased in a self-produced polymeric matrix remain unknown. In this study, using Shewanella oneidensis as a model organism and alginate hydrogel as a model synthetic matrix, we compared the cell growth and protein expression in entrapped cultures and biofilms. The hydrogel-entrapped cultures were found to exhibit a growth rate comparable with biofilms. There was no substantial difference in cell viability, surface charge, as well as hydrophobicity between the cells grown in alginate hydrogel and those grown in biofilms. However, the gel-entrapped cultures were found to be physiologically different from biofilms. The gel-entrapped cultures had a higher demand for metabolic energy. The siderophore-mediated iron uptake was repressed in the gel-entrapped cells. The presence of the hydrogel matrix decreased the expression of proteins involved in biofilm formation, while inducing the production of extracellular DNA (eDNA) in the gel-entrapped cultures. These results advance the fundamental understanding of the physiology of hydrogel-entrapped cells, which can lead to more efficient biofilm mimic-based applications.

Culture of hESC-derived pancreatic progenitors in alginate-based scaffolds.

PubMed

Formo, Kjetil; Cho, Candy H-H; Vallier, Ludovic; Strand, Berit L

2015-12-01

The effect of alginate-based scaffolds with added basement membrane proteins on the in vitro development of hESC-derived pancreatic progenitors was investigated. Cell clusters were encapsulated in scaffolds containing the basement membrane proteins collagen IV, laminin, fibronectin, or extracellular matrix-derived peptides, and maintained in culture for up to 46 days. The cells remained viable throughout the experiment with no signs of central necrosis. Whereas nonencapsulated cells aggregated into larger clusters, some of which showed signs of morphological changes and tissue organization, the alginate matrix stabilized the cluster size and displayed more homogeneous cell morphologies, allowing culture for long periods of time. For all conditions tested, a stable or declining expression of insulin and PDX1 and an increase in glucagon and somatostatin over time indicated a progressive reduction in beta cell-related gene expression. Alginate scaffolds can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors. Although no increase in insulin and PDX1 gene expression after alginate-immobilized cell culture was seen in this study, further optimization of the matrix physicochemical and biological properties and of the medium composition may still be a relevant strategy to promote the stabilization or maturation of stem cell-derived beta cells. © 2015 Wiley Periodicals, Inc.

A Microfluidic Localized, Multiple Cell Culture Array using Vacuum Actuated Cell Seeding: Integrated Anticancer Drug Testing

PubMed Central

Gao, Yan; Li, Peng

2013-01-01

In this study, we introduced a novel and convenient approach to culture multiple cells in localized arrays of microfluidic chambers using one-step vacuum actuation. In one device, we integrated 8 individually addressable regions of culture chambers, each only requiring one simple vacuum operation to seed cells lines. Four cell lines were seeded in designated regions in one device via sequential injection with high purity (99.9%-100%) and cultured for long-term. The on-chip simultaneous culture of HuT 78, Ramos, PC-3 and C166-GFP cells for 48 h was demonstrated with viabilities of 92%+/−2%, 94%+/−4%, 96%+/−2% and 97%+/−2%, respectively. The longest culture period for C166-GFP cells in this study was 168 h with a viability of 96%+/−10%. Cell proliferation in each individual side channel can be tracked. Mass transport between the main channel and side channels was achieved through diffusion and studied using fluorescein solution. The main advantage of this device is the capability to perform multiple cell-based assays on the same device for better comparative studies. After treating cells with staurosporine or anti-human CD95 for 16 h, the apoptotic cell percentage of HuT 78, CCRF-CEM, PC-3 and Ramos cells were 36%+/−3%, 24%+/−4%, 12%+/−2%, 18%+/−4% for staurosporine, and 63%+/−2%, 45%+/−1%, 3%+/−3%, 27%+/−12% for anti-human CD95, respectively. With the advantages of enhanced integration, ease of use and fabrication, and flexibility, this device will be suitable for long-term multiple cell monitoring and cell based assays. PMID:23813077

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

PubMed

Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

2016-02-01

To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

Human cell culture in a space bioreactor

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1988-01-01

Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation.

PubMed

Liu, Yarong; Fox, Victoria; Lei, Yuning; Hu, Biliang; Joo, Kye-Il; Wang, Pin

2014-07-01

The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications. © 2013 Wiley Periodicals, Inc.

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

PubMed Central

Agley, Chibeza C.; Rowlerson, Anthea M.; Velloso, Cristiana P.; Lazarus, Norman L.; Harridge, Stephen D. R.

2015-01-01

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56– and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 106 ± 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56– cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package. PMID:25650991

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization.

PubMed

Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar; Alexander, Dorothea

2017-01-01

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems.

PubMed

Badenes, Sara M; Fernandes, Tiago G; Rodrigues, Carlos A V; Diogo, Maria Margarida; Cabral, Joaquim M S

2016-09-20

Human pluripotent stem cells (hPSC) have attracted a great attention as an unlimited source of cells for cell therapies and other in vitro biomedical applications such as drug screening, toxicology assays and disease modeling. The implementation of scalable culture platforms for the large-scale production of hPSC and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of cells for these applications. Microcarrier technology has been emerging as an effective approach for the large scale ex vivo hPSC expansion and differentiation. This review presents recent achievements in hPSC microcarrier-based culture systems and discusses the crucial aspects that influence the performance of these culture platforms. Recent progress includes addressing chemically-defined culture conditions for manufacturing of hPSC and their derivatives, with the development of xeno-free media and microcarrier coatings to meet good manufacturing practice (GMP) quality requirements. Finally, examples of integrated platforms including hPSC expansion and directed differentiation to specific lineages are also presented in this review. Copyright © 2016 Elsevier B.V. All rights reserved.

Synchrony in human, mouse and bacterial cell cultures--a comparison

NASA Technical Reports Server (NTRS)

Helmstetter, Charles E.; Thornton, Maureen; Romero, Ana; Eward, K. Leigh

2003-01-01

Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.

Glycyrrhiza glabra (Linn.) and Lavandula officinalis (L.) cell suspension cultures-based biotransformation of β-artemether.

PubMed

Patel, Suman; Gaur, Rashmi; Upadhyaya, Mohita; Mathur, Archana; Mathur, Ajay K; Bhakuni, Rajendra S

2011-07-01

The biotransformation of β-artemether (1) by cell suspension cultures of Glycyrrhiza glabra and Lavandula officinalis is reported here for the first time. The major biotransformed product appeared as a grayish-blue color spot on thin-layer chromatography (TLC) with transparent crystal-like texture. Based on its infrared (IR) and (1)H nuclear magnetic resonance (NMR) spectra, the product was characterized as a tetrahydrofuran (THF)-acetate derivative (2). The highest conversion efficiencies of 57 and 60% were obtained when 8-9-day-old cell suspensions of G. glabra and L. officinalis were respectively fed with 4-7 mg of compound 1 in 40 ml of medium per culture and the cells were harvested after 2-5 days of incubation. The addition of compound 1 at the beginning of the culture cycle caused severe growth depression in a dose-dependent manner, resulting in poor bioconversion efficiency of ~25% at 2-5 mg/culture dose only.

Surface Acoustic Waves (SAW)-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures.

PubMed

Wang, Tao; Green, Ryan; Nair, Rajesh Ramakrishnan; Howell, Mark; Mohapatra, Subhra; Guldiken, Rasim; Mohapatra, Shyam Sundar

2015-12-19

Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose-response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell culture models, which may have potential applications in both longitudinal 3D cell cultures in cancer biology and in regenerative medicine.

Continuous microcarrier-based cell culture in a benchtop microfluidic bioreactor.

PubMed

Abeille, F; Mittler, F; Obeid, P; Huet, M; Kermarrec, F; Dolega, M E; Navarro, F; Pouteau, P; Icard, B; Gidrol, X; Agache, V; Picollet-D'hahan, N

2014-09-21

Microfluidic bioreactors are expected to impact cell therapy and biopharmaceutical production due to their ability to control cellular microenvironments. This work presents a novel approach for continuous cell culture in a microfluidic system. Microcarriers (i.e., microbeads) are used as growth support for anchorage-dependent mammalian cells. This approach eases the manipulation of cells within the system and enables harmless extraction of cells. Moreover, the microbioreactor uses a perfusion function based on the biocompatible integration of a porous membrane to continuously feed the cells. The perfusion rate is optimized through simulations to provide a stable biochemical environment. Thermal management is also addressed to ensure a homogeneous bioreactor temperature. Eventually, incubator-free cell cultures of Drosophila S2 and PC3 cells are achieved over the course of a week using this bioreactor. In future applications, a more efficient alternative to harvesting cells from microcarriers is also anticipated as suggested by our positive results from the microcarrier digestion experiments.

Integrated microfluidic devices for combinatorial cell-based assays.

PubMed

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert; Radu, Caius G; Witte, Owen N; Lee, Ki-Bum; Tseng, Hsian-Rong

2009-06-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-microChip), for parallel analyses of the effects of microenvironmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibroblast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-microChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology.

Integrated microfluidic devices for combinatorial cell-based assays

PubMed Central

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert

2010-01-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-μChip), for parallel analyses of the effects of microenvir-onmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibro-blast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-μChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology. PMID:19130244

A fibrin-supported myocardial organ culture for isolation of cardiac stem cells via the recapitulation of cardiac homeostasis.

PubMed

Kim, Jong-Tae; Chung, Hye Jin; Seo, Ji-Yeon; Yang, Young-Il; Choi, Min-Young; Kim, Hyeong-In; Yang, Tae-Hyun; Lee, Won-Jin; Youn, Young Chul; Kim, Hye Jung; Kim, Yeon Mee; Lee, Hyukjin; Jang, Yang-Soo; Lee, Seung-Jin

2015-04-01

There is great interest in the development of cardiac stem cells (CSCs) cell-based therapeutics; thus, clinical translation requires an efficient method for attaining therapeutic quantities of these cells. Furthermore, an in vitro model to investigate the mechanisms regulating the cardiac homeostasis is crucial. We sought to develop a simple myocardial culture method for enabling both the recapitulation of myocardial homeostasis and the simultaneous isolation of CSCs. The intact myocardial fragments were encapsulated 3-dimensionally into the fibrin and cultured under dynamic conditions. The fibrin provided secure physical support and substratum to the myocardium, which mediated integrin-mediated cell signaling that allowed in situ renewal, outgrowth and cardiomyogenic differentiation of CSCs, mimicking myocardial homeostasis. Since our culture maintained the myocardial CSCs niches, it was possible to define the identity of in vitro renewed CSCs that situated in the interstitium between cardiomyocytes and microvessels. Lastly, the use of matrix-restricted fibrinolysis enabled the selective isolation of outgrown CSCs that retained the clonogenicity, long-term growth competency and cardiovascular commitment potential. Collectively, this myocardial culture might be used as an alternative tool for studying cardiac biology and developing cell-based therapeutics. Copyright © 2015 Elsevier Ltd. All rights reserved.

From fibroblasts and stem cells: implications for cell therapies and somatic cloning.

PubMed

Kues, Wilfried A; Carnwath, Joseph W; Niemann, Heiner

2005-01-01

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates.

PubMed

Dowd, Jason E; Jubb, Anthea; Kwok, K Ezra; Piret, James M

2003-05-01

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of approximately 5 x 10(6) cells mL(-1). Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell(-1) day(-1). Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized ( approximately 40 mg L(-1)) at 0.2 nL cell(-1) day(-1). The volumetric protein productivity ( approximately 60 mg L(-1) day(-1) was maximized above 0.3 nL cell(-1) day(-1). The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures.

Cellular automata model for human articular chondrocytes migration, proliferation and cell death: An in vitro validation.

PubMed

Vaca-González, J J; Gutiérrez, M L; Guevara, J M; Garzón-Alvarado, D A

2017-01-01

Articular cartilage is characterized by low cell density of only one cell type, chondrocytes, and has limited self-healing properties. When articular cartilage is affected by traumatic injuries, a therapeutic strategy such as autologous chondrocyte implantation is usually proposed for its treatment. This approach requires in vitro chondrocyte expansion to yield high cell number for cell transplantation. To improve the efficiency of this procedure, it is necessary to assess cell dynamics such as migration, proliferation and cell death during culture. Computational models such as cellular automata can be used to simulate cell dynamics in order to enhance the result of cell culture procedures. This methodology has been implemented for several cell types; however, an experimental validation is required for each one. For this reason, in this research a cellular automata model, based on random-walk theory, was devised in order to predict articular chondrocyte behavior in monolayer culture during cell expansion. Results demonstrated that the cellular automata model corresponded to cell dynamics and computed-accurate quantitative results. Moreover, it was possible to observe that cell dynamics depend on weighted probabilities derived from experimental data and cell behavior varies according to the cell culture period. Thus, depending on whether cells were just seeded or proliferated exponentially, culture time probabilities differed in percentages in the CA model. Furthermore, in the experimental assessment a decreased chondrocyte proliferation was observed along with increased passage number. This approach is expected to having other uses as in enhancing articular cartilage therapies based on tissue engineering and regenerative medicine.

Pericellular oxygen monitoring with integrated sensor chips for reproducible cell culture experiments.

PubMed

Kieninger, J; Aravindalochanan, K; Sandvik, J A; Pettersen, E O; Urban, G A

2014-04-01

Here we present an application, in two tumour cell lines, based on the Sensing Cell Culture Flask system as a cell culture monitoring tool for pericellular oxygen sensing. T-47D (human breast cancer) and T98G (human brain cancer) cells were cultured either in atmospheric air or in a glove-box set at 4% oxygen, in both cases with 5% CO2 in the gas phase. Pericellular oxygen tension was measured with the help of an integrated sensor chip comprising oxygen sensor arrays. Obtained results illustrate variation of pericellular oxygen tension in attached cells covered by stagnant medium. Independent of incubation conditions, low pericellular oxygen concentration levels, usually associated with hypoxia, were found in dense cell cultures. Respiration alone brought pericellular oxygen concentration down to levels which could activate hypoxia-sensing regulatory processes in cultures believed to be aerobic. Cells in culture believed to experience conditions of mild hypoxia may, in reality, experience severe hypoxia. This would lead to incorrect assumptions and suggests that pericellular oxygen concentration readings are of great importance to obtain reproducible results when dealing with hypoxic and normoxic (aerobic) incubation conditions. The Sensing Cell Culture Flask system allows continuous monitoring of pericellular oxygen concentration with outstanding long-term stability and no need for recalibration during cell culture experiments. The sensor is integrated into the flask bottom, thus in direct contact with attached cells. No additional equipment needs to be inserted into the flask during culturing. Transparency of the electrochemical sensor chip allows optical inspection of cells attached on top of the sensor. © 2014 John Wiley & Sons Ltd.

Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

PubMed

Schmelzer, Eva; Finoli, Anthony; Nettleship, Ian; Gerlach, Jörg C

2015-04-01

The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture. © 2015 Wiley Periodicals, Inc.

Erythrocyte Enrichment in Hematopoietic Progenitor Cell Cultures Based on Magnetic Susceptibility of the Hemoglobin

PubMed Central

Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V.; Moore, Lee R.; Chalmers, Jeffrey J.; Zborowski, Maciej

2012-01-01

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. PMID:22952572

Advances in tissue engineering through stem cell-based co-culture.

PubMed

Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

2015-05-01

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals.

PubMed

Sahare, Mahesh G; Suyatno; Imai, Hiroshi

2018-04-01

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans. Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized. In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species-specific requirements for growth factors and mechanisms supporting the self-renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential. Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.

On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect.

PubMed

Xu, Jianfeng; Zhang, Ningning

2014-12-01

Plant cell culture is emerging as an alternative bioproduction system for recombinant pharmaceuticals. Growing plant cells in vitro under controlled environmental conditions allows for precise control over cell growth and protein production, batch-to-batch product consistency and a production process aligned with current good manufacturing practices. With the recent US FDA approval and commercialization of the world's first plant cell-based recombinant pharmaceutical for human use, β-glucocerebrosidase for treatment of Gaucher's disease, a new era has come in which plant cell culture shows high potential to displace some established platform technologies in niche markets. This review updates the progress in plant cell culture processing technology, highlights recent commercial successes and discusses the challenges that must be overcome to make this platform commercially viable.

Kidney cell electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry.

Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

PubMed Central

Ruedel, Anke; Hofmeister, Simone; Bosserhoff, Anja-Katrin

2013-01-01

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies. PMID:24294400

Using digital inline holographic microscopy and quantitative phase contrast imaging to assess viability of cultured mammalian cells

NASA Astrophysics Data System (ADS)

Missan, Sergey; Hrytsenko, Olga

2015-03-01

Digital inline holographic microscopy was used to record holograms of mammalian cells (HEK293, B16, and E0771) in culture. The holograms have been reconstructed using Octopus software (4Deep inwater imaging) and phase shift maps were unwrapped using the FFT-based phase unwrapping algorithm. The unwrapped phase shifts were used to determine the maximum phase shifts in individual cells. Addition of 0.5 mM H2O2 to cell media produced rapid rounding of cultured cells, followed by cell membrane rupture. The cell morphology changes and cell membrane ruptures were detected in real time and were apparent in the unwrapped phase shift images. The results indicate that quantitative phase contrast imaging produced by the digital inline holographic microscope can be used for the label-free real time automated determination of cell viability and confluence in mammalian cell cultures.

The influence of incubation time on adenovirus quantitation in A549 cells by most probable number

EPA Science Inventory

Cell culture based assays used to detect waterborne viruses typically call for incubating the sample for at least two weeks in order to ensure that all the culturable virus present is detected. Historically, this estimate was based, at least in part, on the length of time used fo...

Ex-vivo dynamic 3-D culture of human tissues in the RCCS™ bioreactor allows the study of Multiple Myeloma biology and response to therapy.

PubMed

Ferrarini, Marina; Steimberg, Nathalie; Ponzoni, Maurilio; Belloni, Daniela; Berenzi, Angiola; Girlanda, Stefania; Caligaris-Cappio, Federico; Mazzoleni, Giovanna; Ferrero, Elisabetta

2013-01-01

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.

Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCS™ Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy

PubMed Central

Ponzoni, Maurilio; Belloni, Daniela; Berenzi, Angiola; Girlanda, Stefania; Caligaris-Cappio, Federico; Mazzoleni, Giovanna; Ferrero, Elisabetta

2013-01-01

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy. PMID:23990965

Exponential growth kinetics for Polyporus versicolor and Pleurotus ostreatus in submerged culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carroad, P.A.; Wilke, C.R.

1977-04-01

Simple mathematical models for a batch culture of pellet-forming fungi in submerged culture were tested on growth data for Polyporus versicolor (ATCC 12679) and Pleurotus ostreatus (ATCC 9415). A kinetic model based on a growth rate proportional to the two-thirds power of the cell mass was shown to be satisfactory. A model based on a growth rate directly proportional to the cell mass fitted the data equally well, however, and may be preferable because of mathematical simplicity.

A continuous perfusion microplate for cell culture.

PubMed

Goral, Vasiliy N; Zhou, Chunfeng; Lai, Fang; Yuen, Po Ki

2013-03-21

We describe a 96-well microplate with fluidically connected wells that enables the continuous fluid perfusion between wells without the need for external pumping. A single unit in such a perfusion microplate consists of three wells: a source well, a sample (cell culture) well in the middle and a waste well. Fluid perfusion is achieved using a combination of the hydrostatic pressure generated by different liquid levels in the wells and the fluid wicking through narrow strips of a cellulose membrane connecting the wells. There is an excellent correspondence between the observed perfusion flow dynamics and the flow simulations based on Darcy's Law. Hepatocytes (C3A cells) cultured for 4 days in the perfusion microplate with no media exchange in the cell culture well had the same viability as hepatocytes exposed to a daily exchange of media. EOC 20 cells that require media conditioned by LADMAC cells were shown to be equally viable in the adjacent cell culture well of the perfusion microplate with LADMAC cells cultured in the source well. Tegafur, a prodrug, when added to primary human hepatocytes in the source well, was metabolized into a cytotoxic metabolite that kills colon cancer cells (HCT 116) cultured in the adjacent cell culture well; no toxicity was observed when only medium was in the source well. These results suggest that the perfusion microplate is a useful tool for a variety of cell culture applications with benefits ranging from labor savings to enabling in vivo-like toxicity studies.

Micropatterns of Matrigel for three-dimensional epithelial cultures.

PubMed

Sodunke, Temitope R; Turner, Keneshia K; Caldwell, Sarah A; McBride, Kevin W; Reginato, Mauricio J; Noh, Hongseok Moses

2007-09-01

Three-dimensional (3D) epithelial culture models are widely used to promote a physiologically relevant microenvironment for the study of normal and aberrant epithelial organization. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hindered by the lack of existing platforms for large-scale 3D cellular studies. Current 3D standard culture does not allow for single spheroid or 'acinus' analysis required for high-throughput systems. Here, we present general strategies for creating bulk micropatterns of Matrigel that can be used as a platform for 3D epithelial culture and cell-based assays at the single acinus level. Both buried and free-standing micropatterns of Matrigel were created using modified soft lithography techniques such as microtransfer molding (microTM) and dry lift-off technique. Surface modification of poly(dimethylsiloxane) (PDMS) with oxygen plasma followed by treatment with poly(2-hydroxy-ethylmethacrylate) (poly-HEMA) was sufficient to promote deformation-free release of Matrigel patterns. In addition, a novel dual-layer dry lift-off technique was developed to simultaneously generate patterns of Matrigel and poly-HEMA on a single substrate. We also demonstrate that the micropatterned Matrigel can support 3D culture originating from a single normal human mammary epithelial (MCF-10A) cell or a human breast cancer cell (MDA-MB-231) with comparable phenotypes to standard 3D culture techniques. Culture of normal MCF-10A cells on micropatterned Matrigel resulted in formation of structures with the characteristic apoptosis of centrally located cells and formation of hollow lumens. Moreover, the carcinoma cell line showed their characteristic formation of disorganized invasive cellular clusters, lacking the normal epithelial architecture on micropatterned Matrigel. Hence, micropatterned Matrigel can be used as a 3D epithelial cell-based platform for a wide variety of applications in epithelial and cancer biology, tissue engineering, as well as gene/drug screening technology.

Disarmed by density: A glycolytic break for immunostimulatory dendritic cells?

PubMed

Nasi, Aikaterini; Rethi, Bence

2013-12-01

We observed a cell concentration-dependent differentiation switch among cultured dendritic cells (DCs) triggered by lactic acid, a product of glycolytic metabolism. In particular, while interleukin (IL)-12, IL-23, and tumor necrosis factor α (TNFα)-producing, migratory DCs developed in sparse cultures, IL-10-producing, non-migratory DCs differentiated in dense cultures. This points to a novel opportunity for tailoring DC-based anticancer therapies through metabolism modulation in developing DCs.

Bioreactors to Influence Stem Cell Fate: Augmentation of Mesenchymal Stem Cell Signaling Pathways via Dynamic Culture Systems

PubMed Central

Yeatts, Andrew B.; Choquette, Daniel T.; Fisher, John P.

2012-01-01

Background Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro. Scope of Review This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems. Major Conclusions The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased. General Significance Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. PMID:22705676

Three Dimensional Primary Hepatocyte Culture

NASA Technical Reports Server (NTRS)

Yoffe, Boris

1998-01-01

Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies

PubMed Central

Pedersen, Jenny M.; Shim, Yoo-Sik; Hans, Vaibhav; Phillips, Martin B.; Macdonald, Jeffrey M.; Walker, Glenn; Andersen, Melvin E.; Clewell, Harvey J.; Yoon, Miyoung

2016-01-01

Accurate prediction of metabolism is a significant outstanding challenge in toxicology. The best predictions are based on experimental data from in vitro systems using primary hepatocytes. The predictivity of the primary hepatocyte-based culture systems, however, is still limited due to well-known phenotypic instability and rapid decline of metabolic competence within a few hours. Dynamic flow bioreactors for three-dimensional cell cultures are thought to be better at recapitulating tissue microenvironments and show potential to improve in vivo extrapolations of chemical or drug toxicity based on in vitro test results. These more physiologically relevant culture systems hold potential for extending metabolic competence of primary hepatocyte cultures as well. In this investigation, we used computational fluid dynamics to determine the optimal design of a flow-based hepatocyte culture system for evaluating chemical metabolism in vitro. The main design goals were (1) minimization of shear stress experienced by the cells to maximize viability, (2) rapid establishment of a uniform distribution of test compound in the chamber, and (3) delivery of sufficient oxygen to cells to support aerobic respiration. Two commercially available flow devices – RealBio® and QuasiVivo® (QV) – and a custom developed fluidized bed bioreactor were simulated, and turbulence, flow characteristics, test compound distribution, oxygen distribution, and cellular oxygen consumption were analyzed. Experimental results from the bioreactors were used to validate the simulation results. Our results indicate that maintaining adequate oxygen supply is the most important factor to the long-term viability of liver bioreactor cultures. Cell density and system flow patterns were the major determinants of local oxygen concentrations. The experimental results closely corresponded to the in silico predictions. Of the three bioreactors examined in this study, we were able to optimize the experimental conditions for long-term hepatocyte cell culture using the QV bioreactor. This system facilitated the use of low system volumes coupled with higher flow rates. This design supports cellular respiration by increasing oxygen concentrations in the vicinity of the cells and facilitates long-term kinetic studies of low clearance test compounds. These two goals were achieved while simultaneously keeping the shear stress experienced by the cells within acceptable limits. PMID:27747210

A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

PubMed

Mollet, Björne B; Bogaerts, Iven L J; van Almen, Geert C; Dankers, Patricia Y W

2017-06-01

Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Live single-cell laser tag.

PubMed

Binan, Loïc; Mazzaferri, Javier; Choquet, Karine; Lorenzo, Louis-Etienne; Wang, Yu Chang; Affar, El Bachir; De Koninck, Yves; Ragoussis, Jiannis; Kleinman, Claudia L; Costantino, Santiago

2016-05-20

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types.

Liver-specific gene expression in cultured human hematopoietic stem cells.

PubMed

Fiegel, Henning C; Lioznov, Michael V; Cortes-Dericks, Lourdes; Lange, Claudia; Kluth, Dietrich; Fehse, Boris; Zander, Axel R

2003-01-01

Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34(+) bone marrow cells to differentiate into hepatocytic cells in vitro. CD34(+) and CD34(-) human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34(+) cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34(+) cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34(-) cells did not show liver-specific gene expression. The results indicate that CD34(+) adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

PubMed

Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

2015-11-01

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd.

Rapid detection of Naegleria fowleri in water distribution pipeline biofilms and drinking water samples.

PubMed

Puzon, Geoffrey J; Lancaster, James A; Wylie, Jason T; Plumb, Iason J

2009-09-01

Rapid detection of pathogenic Naegleria fowler in water distribution networks is critical for water utilities. Current detection methods rely on sampling drinking water followed by culturing and molecular identification of purified strains. This culture-based method takes an extended amount of time (days), detects both nonpathogenic and pathogenic species, and does not account for N. fowleri cells associated with pipe wall biofilms. In this study, a total DNA extraction technique coupled with a real-time PCR method using primers specific for N. fowleri was developed and validated. The method readily detected N. fowleri without preculturing with the lowest detection limit for N. fowleri cells spiked in biofilm being one cell (66% detection rate) and five cells (100% detection rate). For drinking water, the detection limit was five cells (66% detection rate) and 10 cells (100% detection rate). By comparison, culture-based methods were less sensitive for detection of cells spiked into both biofilm (66% detection for <10 cells) and drinking water (0% detection for <10 cells). In mixed cultures of N. fowleri and nonpathogenic Naegleria, the method identified N. fowleri in 100% of all replicates, whereastests with the current consensus primers detected N. fowleri in only 5% of all replicates. Application of the new method to drinking water and pipe wall biofilm samples obtained from a distribution network enabled the detection of N. fowleri in under 6 h, versus 3+ daysforthe culture based method. Further, comparison of the real-time PCR data from the field samples and the standard curves enabled an approximation of N. fowleri cells in the biofilm and drinking water. The use of such a method will further aid water utilities in detecting and managing the persistence of N. fowleri in water distribution networks.

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

PubMed

Kino-Oka, Masahiro; Maeda, Yoshikatsu; Yamamoto, Takeyuki; Sugawara, Katsura; Taya, Masahito

2005-03-01

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

PubMed

Dalley, Andrew J; AbdulMajeed, Ahmad A; Upton, Zee; Farah, Camile S

2013-01-01

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. © 2012 John Wiley & Sons A/S.

Comparison of six different methods to calculate cell densities.

PubMed

Camacho-Fernández, Carolina; Hervás, David; Rivas-Sendra, Alba; Marín, Mª Pilar; Seguí-Simarro, Jose M

2018-01-01

For in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density. A typical example of this is isolated microspore culture, where specific cell densities have been determined for different species. Out of these ranges, microspore growth is not induced, or is severely reduced. A similar situation occurs in many other plant and animal cell culture systems. Traditionally, researchers have used counting chambers (hemacytometers) to calculate cell densities, but little is still known about their technical advantages. In addition, much less information is available about other, alternative methods. In this work, using isolated eggplant microspore cultures and fluorescent beads (fluorospheres) as experimental systems, we performed a comprehensive comparison of six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC). Our results show that from a technical perspective, hemacytometers are the most reasonable option for cell counting, which may explain their widely spread use. Automated cell counters represent a good compromise between precision and affordability, although with limited accuracy. Finally, the methods based on flow cytometry were, by far, the best in terms of reproducibility and agreement between them, but they showed deficient accuracy and precision. Together, our results show a thorough technical evaluation of each counting method, provide unambiguous arguments to decide which one is the most convenient for the particular case of each laboratory, and in general, shed light into the best way to determine cell densities for in vitro cell cultures. They may have an impact in such a practice not only in the context of microspore culture, but also in any other plant cell culture procedure, or in any process involving particle counting.

PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination.

PubMed

Garinot, Marie; Fiévez, Virginie; Pourcelle, Vincent; Stoffelbach, François; des Rieux, Anne; Plapied, Laurence; Theate, Ivan; Freichels, Hélène; Jérôme, Christine; Marchand-Brynaert, Jacqueline; Schneider, Yves-Jacques; Préat, Véronique

2007-07-31

To improve the efficiency of orally delivered vaccines, PEGylated PLGA-based nanoparticles displaying RGD molecules at their surface were designed to target human M cells. RGD grafting was performed by an original method called "photografting" which covalently linked RGD peptides mainly on the PEG moiety of the PCL-PEG, included in the formulation. First, three non-targeted formulations with size and zeta potential adapted to M cell uptake and stable in gastro-intestinal fluids, were developed. Their transport by an in vitro model of the human Follicle associated epithelium (co-cultures) was largely increased as compared to mono-cultures (Caco-2 cells). RGD-labelling of nanoparticles significantly increased their transport by co-cultures, due to interactions between the RGD ligand and the beta(1) intregrins detected at the apical surface of co-cultures. In vivo studies demonstrated that RGD-labelled nanoparticles particularly concentrated in M cells. Finally, ovalbumin-loaded nanoparticles were orally administrated to mice and induced an IgG response, attesting antigen ability to elicit an immune response after oral delivery.

In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

PubMed

Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

2014-09-01

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

The third dimension bridges the gap between cell culture and live tissue.

PubMed

Pampaloni, Francesco; Reynaud, Emmanuel G; Stelzer, Ernst H K

2007-10-01

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.

Bioengineered 3D Scaffolds in Cancer Research: Focus on Epithelial to Mesenchymal Transition and Drug Screening.

PubMed

Xu, Xiaoli; Tang, LiLing

2017-01-01

The living environment of cancer cells is complicated and information-rich. Thus, traditional 2D culture mold in vitro cannot mimic the microenvironment of cancer cells exactly. Currently, bioengineered 3D scaffolds have been developed which can better simulate the microenvironment of tumors and fill the gap between 2D culture and clinical application. In this review, we discuss the scaffold materials used for fabrication techniques, biological behaviors of cancer cells in 3D scaffolds and the scaffold-based drug screening. A major emphasis is placed on the description of scaffold-based epithelial to mesenchymal transition and drug screening in 3D culture. By overcoming the defects of traditional 2D culture, 3D scaffolds culture can provide a simpler, safer and more reliable approach for cancer research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

[Determination of DNA content in individual Yersinia pestis cells by using flow cytofluorimetry method: comparative analysis of inhomogeneity in cultures of strains with various biological properties].

PubMed

Kravtsov, A L; Liapin, M N; Shmel'kova, T P; Golovko, E M; Maliukova, T A; Kostiukova, T A; Ezhov, I N

2011-01-01

Comparative analysis of Yersinia pestis strains with various biological properties by DNA content in individual cells. Virulent strain 231, avirulent strain KM 260 (12) [231], that is its isogenic (no-plasmid) derivative, and vaccine strain EV NIIEG were used. 48-hour agar cultures of the studied strains reproduced at 28 degrees C and their subcultures obtained by cultivation of the initial cultures by aeration on liquid nutrient medium from 37 degrees C were prepared. DNA of the fixed bacteria was dyed by a mixture of ethidium bromide and mitramycin, and then the bacteria were studied by using flow cytofluorimeter for the determination of rates of cells with relatively low or high DNA content in the studied bacterial populations. The degree of inhomogeneity of a bacterial population was evaluated by DNA histogram variation coefficient value. In 6 hours of growth at 37 degrees C in optically non-dense bacterial cultures a high degree of DNA content per cell inhomogeneity was established that is related to the activation of DNA replication process in bacteria. In 48 hours of growth this inhomogeneity completely disappeared in the virulent strain cultures and remained in the avirulent strain cultures of the plague pathogen. Based on the studied parameters the vaccine strain held an intermediate position. Further studies of the plague culture DNA content per cell inhomogeneity may become a base for the operative strain differentiation based on pathogenicity level (hazard) for humans, and therefore the requirements for the management of safe working conditions with this microorganism.

Biological Response of Osteoblastic and Chondrogenic Cells to Graphene-Containing PCL/Bioactive Glass Bilayered Scaffolds for Osteochondral Tissue Engineering Applications.

PubMed

Deliormanlı, Aylin M; Atmaca, Harika

2018-05-25

Graphene-containing 13-93 bioactive glass and poly(ε-caprolactone)-based bilayer, electrically conductive scaffolds were prepared for osteochondral tissue repair. Biological response of osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells to the composite scaffolds was assessed under mono-culture and co-culture conditions. Cytotoxicity was investigated using MTT assay, cartilage matrix production was evaluated by Alcian blue staining, and mineralization of both types of cells in the different culture systems was observed by Alizarin red S staining. Results showed that osteoblastic and chondrogenic cells utilized in the study did not show toxic response to the prepared scaffolds under mono-culture conditions and higher cell viability rates were obtained in co-culture conditions. Larger mineralized areas were determined under co-culture conditions and calcium deposition amount significantly increased compared with that in control group samples after 21 days. Additionally, the amount of glycosaminoglycans synthesized in co-culture was higher compared to mono-culture conditions. Electric stimulation applied under mono-culture conditions suppressed the viability of MC3T3-E1 cells whereas it enhanced the viability rates of ATDC5 cells. The study suggests that the designed bilayered osteochondral constructs have the potential for osteochondral defect repair.

Immunogenicity is preferentially induced in sparse dendritic cell cultures

PubMed Central

Nasi, Aikaterini; Bollampalli, Vishnu Priya; Sun, Meng; Chen, Yang; Amu, Sylvie; Nylén, Susanne; Eidsmo, Liv; Rothfuchs, Antonio Gigliotti; Réthi, Bence

2017-01-01

We have previously shown that human monocyte-derived dendritic cells (DCs) acquired different characteristics in dense or sparse cell cultures. Sparsity promoted the development of IL-12 producing migratory DCs, whereas dense cultures increased IL-10 production. Here we analysed whether the density-dependent endogenous breaks could modulate DC-based vaccines. Using murine bone marrow-derived DC models we show that sparse cultures were essential to achieve several key functions required for immunogenic DC vaccines, including mobility to draining lymph nodes, recruitment and massive proliferation of antigen-specific CD4+ T cells, in addition to their TH1 polarization. Transcription analyses confirmed higher commitment in sparse cultures towards T cell activation, whereas DCs obtained from dense cultures up-regulated immunosuppressive pathway components and genes suggesting higher differentiation plasticity towards osteoclasts. Interestingly, we detected a striking up-regulation of fatty acid and cholesterol biosynthesis pathways in sparse cultures, suggesting an important link between DC immunogenicity and lipid homeostasis regulation. PMID:28276533

Genome Editing of Erythroid Cell Culture Model Systems.

PubMed

Yik, Jinfen J; Crossley, Merlin; Quinlan, Kate G R

2018-01-01

Genome editing to introduce specific mutations or to knock out genes in model cell systems has become an efficient platform for research in the fields of molecular biology, genetics, and cell biology. With recent rapid improvements in genome editing techniques, bench-top manipulation of the genome in cell culture has become progressively easier. The application of this knowledge to erythroid cell culture systems now allows the rapid analysis of the downstream effects of virtually any engineered gene disruption or modification in cell systems. Here, we describe a CRISPR/Cas9-based approach to making genomic modifications in erythroid lineage cells which we have successfully used in both murine (MEL) and human (K562) erythroleukaemia immortalized cell lines.

A Novel 3 Dimensional Stromal-based Model for In Vitro Chemotherapy Sensitivity Testing of Leukemia Cells

PubMed Central

Aljitawi, Omar S.; Li, Dandan; Xiao, Yinghua; Zhang, Da; Ramachandran, Karthik; Stehno-Bittel, Lisa; Van Veldhuizen, Peter; Lin, Tara L.; Kambhampati, Suman; Garimella, Rama

2014-01-01

The disparate responses of leukemia cells to chemotherapy in vivo, compared to in vitro, is partly related to the interactions of leukemic cells and the 3 dimensional (3D) bone marrow stromal microenvironment. We investigated the effects of chemotherapy agents on leukemic cell lines co-cultured with human bone marrow mesenchymal stem cell (hu-BM-MSC) in 3D. Comparison was made to leukemic cells treated in suspension, or grown on a hu-BM-MSC monolayer (2D conditions). We demonstrated that leukemic cells cultured in 3D were more resistant to drug-induced apoptosis compared to cells cultured in 2D or in suspension. We also demonstrated significant differences in leukemic cell response to chemotherapy using different leukemic cell lines cultured in 3D. We suggest that the differential responses to chemotherapy in 3D may be related to the expression of N-cadherin in the co-culture system. This unique model provides an opportunity to study leukemic cell responses to chemotherapy in 3D. PMID:23566162

Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

PubMed Central

Nakagawa, Yukari; Maeda, Hideko; Murai, Toshimi

2002-01-01

The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I ·  C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I · C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans. PMID:11986265

Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning

PubMed Central

Langhans, Sigrid A.

2018-01-01

Drug development is a lengthy and costly process that proceeds through several stages from target identification to lead discovery and optimization, preclinical validation and clinical trials culminating in approval for clinical use. An important step in this process is high-throughput screening (HTS) of small compound libraries for lead identification. Currently, the majority of cell-based HTS is being carried out on cultured cells propagated in two-dimensions (2D) on plastic surfaces optimized for tissue culture. At the same time, compelling evidence suggests that cells cultured in these non-physiological conditions are not representative of cells residing in the complex microenvironment of a tissue. This discrepancy is thought to be a significant contributor to the high failure rate in drug discovery, where only a low percentage of drugs investigated ever make it through the gamut of testing and approval to the market. Thus, three-dimensional (3D) cell culture technologies that more closely resemble in vivo cell environments are now being pursued with intensity as they are expected to accommodate better precision in drug discovery. Here we will review common approaches to 3D culture, discuss the significance of 3D cultures in drug resistance and drug repositioning and address some of the challenges of applying 3D cell cultures to high-throughput drug discovery. PMID:29410625

A phenotypic screening approach to identify anticancer compounds derived from marine fungi.

PubMed

Ellinger, Bernhard; Silber, Johanna; Prashar, Anjali; Landskron, Johannes; Weber, Jonas; Rehermann, Sarah; Müller, Franz-Josef; Smith, Stephen; Wrigley, Stephen; Taskén, Kjetil; Gribbon, Philip; Labes, Antje; Imhoff, Johannes F

2014-04-01

This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner.

Putting the Spotlight Back on Plant Suspension Cultures

PubMed Central

Santos, Rita B.; Abranches, Rita; Fischer, Rainer; Sack, Markus; Holland, Tanja

2016-01-01

Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®)—the first licensed recombinant pharmaceutical protein derived from plants—is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plant cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models, and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon. PMID:27014320

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology;more » CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.« less

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

PubMed

Spaethling, Jennifer M; Na, Young-Ji; Lee, Jaehee; Ulyanova, Alexandra V; Baltuch, Gordon H; Bell, Thomas J; Brem, Steven; Chen, H Isaac; Dueck, Hannah; Fisher, Stephen A; Garcia, Marcela P; Khaladkar, Mugdha; Kung, David K; Lucas, Timothy H; O'Rourke, Donald M; Stefanik, Derek; Wang, Jinhui; Wolf, John A; Bartfai, Tamas; Grady, M Sean; Sul, Jai-Yoon; Kim, Junhyong; Eberwine, James H

2017-01-17

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

PubMed

Chen, Kevin G; Mallon, Barbara S; Johnson, Kory R; Hamilton, Rebecca S; McKay, Ronald D G; Robey, Pamela G

2014-05-01

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications. Published by Elsevier B.V.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

PubMed Central

Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

2010-01-01

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

Easy-to-use microfluidic chip for long-term 3D-cell cultures

NASA Astrophysics Data System (ADS)

Bunge, Frank; van den Driesche, Sander; Vellekoop, Michael J.

2017-05-01

We present a microfluidic chip for an easy setup of a 3D-culture of mammalian cells. The chip contains feeding structures and gas supply for long-term cultivation of mammalian cells. The device is fabricated out of hard materials like silicon and glass that are all highly biocompatible. The chip uses the concept of surficial phaseguides that allows the partial filling of a microfluidic chip with liquids based on hydrophobic and hydrophilic surfaces. Here, a suspension of mammalian cells and melted agarose is filled into the chip and is pulled by the capillary pressure on the hydrophilic areas but not on the hydrophobic phaseguides. Consequently, only a part of the chip is filled with the agarose which gels by cooling a form the 3D-cell culture. The unfilled areas are used as supply structures for nutrition and gases. So the supply is based on diffusion and the supply of nutrition and gases is controlled independently. We cultured HaCaT-cells over 24 hours in our device and achieve a good viability.

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

PubMed Central

Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar

2017-01-01

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells. PMID:29391870

A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

PubMed

Koban, Robert; Neumann, Markus; Daugs, Aila; Bloch, Oliver; Nitsche, Andreas; Langhammer, Stefan; Ellerbrok, Heinz

2018-02-01

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

Clinical-Grade Manufacturing of Therapeutic Human Mesenchymal Stem/Stromal Cells in Microcarrier-Based Culture Systems.

PubMed

Fernandes-Platzgummer, Ana; Carmelo, Joana G; da Silva, Cláudia Lobato; Cabral, Joaquim M S

2016-01-01

The therapeutic potential of mesenchymal stem/stromal cells (MSC) has triggered the need for high cell doses in a vast number of clinical applications. This demand requires the development of good manufacturing practices (GMP)-compliant ex vivo expansion protocols that should be effective to deliver a robust and reproducible supply of clinical-grade cells in a safe and cost-effective manner. Controlled stirred-tank bioreactor systems under xenogeneic (xeno)-free culture conditions offer ideal settings to develop and optimize cell manufacturing to meet the standards and needs of human MSC for cellular therapies. Herein we describe two microcarrier-based stirred culture systems using spinner flasks and controlled stirred-tank bioreactors under xeno-free conditions for the efficient ex vivo expansion of human bone marrow and adipose tissue-derived MSC.

A hanging drop culture method to study terminal erythroid differentiation.

PubMed

Gutiérrez, Laura; Lindeboom, Fokke; Ferreira, Rita; Drissen, Roy; Grosveld, Frank; Whyatt, David; Philipsen, Sjaak

2005-10-01

To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation. Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation. We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells. The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations.

Importance of a diffusion-dominant small volume to activate cell-secreted soluble factor signaling in embryonic stem cell culture in microbioreactors: a mathematical model based study.

PubMed

Chowdhury, Mohammad Mahfuz; Fujii, Teruo; Sakai, Yasuyuki

2013-07-01

In our previous studies, we observed that cell-secreted BMP4 had a prominent influence on mouse embryonic stem cell (mESC) behaviors in a membrane-based two-chambered microbioreactor (MB), but not in a macro-scale culture (6-well plate/6WP). In this study, we investigated how the physical aspects of these cultures regulated BMP4 signaling by developing mathematical models of the cultures. The models estimated signaling activity in the cultures by considering size of the undifferentiated mESC colonies and their growth, diffusion of BMP4, and BMP4 trafficking process in the colonies. The models successfully depicted measured profile of BMP4 concentration in the culture medium which was two times higher in the MB than that in the 6WP during 5-day culture. The models estimated that, owing to the small volume and the membrane, cells were exposed to a higher BMP4 concentration in the top chamber of the MB than that in the 6WP culture. The higher concentration of BMP4 induced a higher concentration of BMP4-bound receptor in the colony in the MB than in the 6WP, thereby leading to the higher activation of BMP4 signaling in the MB. The models also predicted that the size of the MB, but not that of the 6WP, was suitable for maximizing BMP4 accumulation and upregulating its signaling. This study will be helpful in analyzing culture systems, designing microfluidic devices for controlling ESC or other cell behavior. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect

PubMed Central

Xu, Jianfeng; Zhang, Ningning

2014-01-01

Plant cell culture is emerging as an alternative bioproduction system for recombinant pharmaceuticals. Growing plant cells in vitro under controlled environmental conditions allows for precise control over cell growth and protein production, batch-to-batch product consistency and a production process aligned with current good manufacturing practices. With the recent US FDA approval and commercialization of the world’s first plant cell-based recombinant pharmaceutical for human use, β-glucocerebrosidase for treatment of Gaucher’s disease, a new era has come in which plant cell culture shows high potential to displace some established platform technologies in niche markets. This review updates the progress in plant cell culture processing technology, highlights recent commercial successes and discusses the challenges that must be overcome to make this platform commercially viable. PMID:25621170

Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine.

PubMed

Lobo, Nazleen C; Gedye, Craig; Apostoli, Anthony J; Brown, Kevin R; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A S; Ailles, Laurie

2016-07-16

Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability to establish primary cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients.

Three-Dimensional Magnetic Levitation Culture System Simulating White Adipose Tissue.

PubMed

Tseng, Hubert; Daquinag, Alexes C; Souza, Glauco R; Kolonin, Mikhail G

2018-01-01

White adipose tissue (WAT) has attracted interest for tissue engineering and cell-based therapies as an abundant source of adipose stem/stromal cells (ASC). However, technical challenges in WAT cell culture have limited its applications in regenerative medicine. Traditional two-dimensional (2D) cell culture models, which are essentially monolayers of cells on glass or plastic substrates, inadequately represent tissue architecture, biochemical concentration gradients, substrate stiffness, and most importantly for WAT research, cell phenotypic heterogeneity. Physiological cell culture platforms for WAT modeling must recapitulate the native diversity of cell types and their coordination within the organ. For this purpose, we developed a three-dimensional (3D) model using magnetic levitation. Here, we describe our protocol that we successfully employed to build adipose tissue organoids (adipospheres) that preserve the heterogeneity of the constituent cell types in vitro. We demonstrate the capacity of assembling adipospheres from multiple cell types, including ASCs, endohtelial cells, and leukocytes that recreate tissue organization. These adipospheres mimicked WAT organogenesis in that they enabled the formation of vessel-like endothelial structures with lumens and differentiation of unilocular adipocytes. Altogether, magnetic levitation is a cell culture platform that recreates tissue structure, function, and heterogeneity in vitro, and serves as a foundation for high-throughput WAT tissue culture and analysis.

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

PubMed Central

Dargaville, Tim R; Hollier, Brett G; Shokoohmand, Ali; Hoogenboom, Richard

2014-01-01

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG). PMID:24714592

Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications

NASA Astrophysics Data System (ADS)

Clements, Isaac P.; Millard, Daniel C.; Nicolini, Anthony M.; Preyer, Amanda J.; Grier, Robert; Heckerling, Andrew; Blum, Richard A.; Tyler, Phillip; McSweeney, K. M.; Lu, Yi-Fan; Hall, Diana; Ross, James D.

2016-03-01

Microelectrode array (MEA) technology enables advanced drug screening and "disease-in-a-dish" modeling by measuring the electrical activity of cultured networks of neural or cardiac cells. Recent developments in human stem cell technologies, advancements in genetic models, and regulatory initiatives for drug screening have increased the demand for MEA-based assays. In response, Axion Biosystems previously developed a multiwell MEA platform, providing up to 96 MEA culture wells arrayed into a standard microplate format. Multiwell MEA-based assays would be further enhanced by optogenetic stimulation, which enables selective excitation and inhibition of targeted cell types. This capability for selective control over cell culture states would allow finer pacing and probing of cell networks for more reliable and complete characterization of complex network dynamics. Here we describe a system for independent optogenetic stimulation of each well of a 48-well MEA plate. The system enables finely graded control of light delivery during simultaneous recording of network activity in each well. Using human induced pluripotent stem cell (hiPSC) derived cardiomyocytes and rodent primary neuronal cultures, we demonstrate high channel-count light-based excitation and suppression in several proof-of-concept experimental models. Our findings demonstrate advantages of combining multiwell optical stimulation and MEA recording for applications including cardiac safety screening, neural toxicity assessment, and advanced characterization of complex neuronal diseases.

Specimen Sample Preservation for Cell and Tissue Cultures

NASA Technical Reports Server (NTRS)

Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

1996-01-01

The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

A Substance Exchanger-Based Bioreactor Culture of Pig Discs for Studying the Immature Nucleus Pulposus.

PubMed

Li, Pei; Gan, Yibo; Wang, Haoming; Xu, Yuan; Song, Lei; Wang, Liyuan; Ouyang, Bin; Zhou, Qiang

2017-11-01

Various research models have been developed to study the biology of disc cells. Recently, the adult disc nucleus pulposus (NP) has been well studied. However, the immature NP is underinvestigated due to a lack of a suitable model. This study aimed to establish an organ culture of immature porcine disc by optimizing culture conditions and using a self-developed substance exchanger-based bioreactor. Immature porcine discs were first cultured in the bioreactor for 7 days at various levels of glucose (low, medium, high), osmolarity (hypo-, iso-, hyper-) and serum (5, 10, 20%) to determine the respective optimal level. The porcine discs were then cultured under the optimized conditions in the novel bioreactor, and were compared with fresh discs at day 14. For high-glucose, iso-osmolarity, or 10% serum, cell viability, the gene expression profile (for anabolic genes and catabolic genes), and glycosaminoglycan (GAG) and hydroxyproline (HYP) contents were more favorable than for other levels of glucose, osmolarity, and serum. When the immature discs were cultured under the optimized conditions using the novel bioreactor for 14 days, the viability of the immature NP was maintained based on histology, cell viability, GAG and HYP contents, and matrix molecule expression. In conclusion, the viability of the immature NP in organ culture could be maintained under the optimized culture conditions (high-glucose, iso-osmolarity, and 10% serum) in the substance exchanger-based bioreactor. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

On-chip immune cell activation and subsequent time-resolved magnetic bead-based cytokine detection.

PubMed

Kongsuphol, Patthara; Liu, Yunxiao; Ramadan, Qasem

2016-10-01

Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of triple-layered microfluidic chambers was fabricated to enable simultaneous cell culture under perfusion flow and detection of the induced cytokines at multiple time-points. Each culture chamber comprises three fluidic compartments which are dedicated to, cell culture, perfusion and immunoassay. The three compartments are separated by porous membranes, which allow the diffusion of fresh nutrient from the perfusion compartment into the cell culture compartment and cytokines secretion from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture and without minimizing benefit gain from perfusion. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The cytokines released from differential stimuli were quantified in situ in non-differentiated U937 monocytes and differentiated macrophages.

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

PubMed

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

2014-04-15

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

On-line sequential injection-capillary electrophoresis for near-real-time monitoring of extracellular lactate in cell culture flasks.

PubMed

Alhusban, Ala A; Gaudry, Adam J; Breadmore, Michael C; Gueven, Nuri; Guijt, Rosanne M

2014-01-03

Cell culture has replaced many in vivo studies because of ethical and regulatory measures as well as the possibility of increased throughput. Analytical assays to determine (bio)chemical changes are often based on end-point measurements rather than on a series of sequential determinations. The purpose of this work is to develop an analytical system for monitoring cell culture based on sequential injection-capillary electrophoresis (SI-CE) with capacitively coupled contactless conductivity detection (C(4)D). The system was applied for monitoring lactate production, an important metabolic indicator, during mammalian cell culture. Using a background electrolyte consisting of 25mM tris(hydroxymethyl)aminomethane, 35mM cyclohexyl-2-aminoethanesulfonic acid with 0.02% poly(ethyleneimine) (PEI) at pH 8.65 and a multilayer polymer coated capillary, lactate could be resolved from other compounds present in media with relative standard deviations 0.07% for intraday electrophoretic mobility and an analysis time of less than 10min. Using the human embryonic kidney cell line HEK293, lactate concentrations in the cell culture medium were measured every 20min over 3 days, requiring only 8.73μL of sample per run. Combining simplicity, portability, automation, high sample throughput, low limits of detection, low sample consumption and the ability to up- and outscale, this new methodology represents a promising technique for near real-time monitoring of chemical changes in diverse cell culture applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel.

PubMed

Trojani, Christophe; Weiss, Pierre; Michiels, Jean-François; Vinatier, Claire; Guicheux, Jérôme; Daculsi, Guy; Gaudray, Patrick; Carle, Georges F; Rochet, Nathalie

2005-09-01

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

PubMed

Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

2016-01-01

As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

The expression of cancer stem cell markers in human colorectal carcinoma cells in a microenvironment dependent manner.

PubMed

Stankevicius, Vaidotas; Kunigenas, Linas; Stankunas, Edvinas; Kuodyte, Karolina; Strainiene, Egle; Cicenas, Jonas; Samalavicius, Narimantas E; Suziedelis, Kestutis

2017-03-18

Numerous lines of evidence support the hierarchical model of cancer development and tumor initiation. According to the theory, cancer stem cells play a crucial role in the formation of the tumor and should be targeted for more effective anticancer treatment. However, cancer stem cells quickly loose their characteristics when propagated as 2D cell culture, indicating that the 2D cell culture does not provide the appropriate settings to maintain an in vivo environment. In this study we have investigated the expression of self-renewal, cancer stem cell and epithelial to mesenchymal transition markers after the transfer of human colorectal carcinoma cell DLD1 and HT29 lines from 2D cell cultures to scaffold-attached laminin rich extracellular matrix and scaffold-free multicellular spheroid 3D culture models. Based on the up-regulated expression of multipotency, CSC and EMT markers, our data suggests that human colorectal carcinoma cells grown in 3D exhibit enhanced cancer stem cell characteristics. Therefore, in order to design more efficient targeted therapies, we suggest that 3D cell culture models should be employed in cancer stem cell research. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Developing global regression models for metabolite concentration prediction regardless of cell line.

PubMed

André, Silvère; Lagresle, Sylvain; Da Sliva, Anthony; Heimendinger, Pierre; Hannas, Zahia; Calvosa, Éric; Duponchel, Ludovic

2017-11-01

Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells, and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell line in order to obtain accurate models. Biotechnol. Bioeng. 2017;114: 2550-2559. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System.

PubMed

Conway, Michael K; Gerger, Michael J; Balay, Erin E; O'Connell, Rachel; Hanson, Seth; Daily, Neil J; Wakatsuki, Tetsuro

2015-05-14

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Polyelectrolyte Multilayer-Treated Electrodes for Real-Time Electronic Sensing of Cell Proliferation

PubMed Central

Mijares, Geraldine I.; Reyes, Darwin R.; Geist, Jon; Gaitan, Michael; Polk, Brian J.; DeVoe, Don L.

2010-01-01

We report on the use of polyelectrolyte multilayer (PEM) coatings as a non-biological surface preparation to facilitate uniform cell attachment and growth on patterned thin-film gold (Au) electrodes on glass for impedance-based measurements. Extracellular matrix (ECM) proteins are commonly utilized as cell adhesion promoters for electrodes; however, they exhibit degradation over time, thereby imposing limitations on the duration of conductance-based biosensor experiments. The motivation for the use of PEM coatings arises from their long-term surface stability as promoters for cell attachment, patterning, and culture. In this work, a cell proliferation monitoring device was fabricated. It consisted of thin-film Au electrodes deposited with a titanium-tungsten (TiW) adhesion layer that were patterned on a glass substrate and passivated to create active electrode areas. The electrode surfaces were then treated with a poly(ethyleneimine) (PEI) anchoring layer and subsequent bilayers of sodium poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). NIH-3T3 mouse embryonic fibroblast cells were cultured on the device, observed by optical microscopy, and showed uniform growth characteristics similar to those observed on a traditional polystyrene cell culture dish. The optical observations were correlated to electrical measurements on the PEM-treated electrodes, which exhibited a rise in impedance with cell proliferation and stabilized to an approximate 15 % increase as the culture approached confluency. In conclusion, cells proliferate uniformly over gold and glass PEM-treated surfaces, making them useful for continuous impedance-based, real-time monitoring of cell proliferation and for the determination of cell growth rate in cellular assays. PMID:27134780

Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells.

PubMed

Wakui, Takashi; Matsumoto, Tsuyoshi; Matsubara, Kenta; Kawasaki, Tomoyuki; Yamaguchi, Hiroshi; Akutsu, Hidenori

2017-10-01

We propose an image analysis method for quality evaluation of human pluripotent stem cells based on biologically interpretable features. It is important to maintain the undifferentiated state of induced pluripotent stem cells (iPSCs) while culturing the cells during propagation. Cell culture experts visually select good quality cells exhibiting the morphological features characteristic of undifferentiated cells. Experts have empirically determined that these features comprise prominent and abundant nucleoli, less intercellular spacing, and fewer differentiating cellular nuclei. We quantified these features based on experts' visual inspection of phase contrast images of iPSCs and found that these features are effective for evaluating iPSC quality. We then developed an iPSC quality evaluation method using an image analysis technique. The method allowed accurate classification, equivalent to visual inspection by experts, of three iPSC cell lines.

Mimicking Metastases Including Tumor Stroma: A New Technique to Generate a Three-Dimensional Colorectal Cancer Model Based on a Biological Decellularized Intestinal Scaffold.

PubMed

Nietzer, Sarah; Baur, Florentin; Sieber, Stefan; Hansmann, Jan; Schwarz, Thomas; Stoffer, Carolin; Häfner, Heide; Gasser, Martin; Waaga-Gasser, Ana Maria; Walles, Heike; Dandekar, Gudrun

2016-07-01

Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of β-catenin in a subset of cells. One central drawback of 2D cultures-especially in consideration of drug testing-is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context-especially for substances targeting tumor-stroma interactions.

Portable real-time fluorescence cytometry of microscale cell culture analog devices

NASA Astrophysics Data System (ADS)

Kim, Donghyun; Tatosian, Daniel A.; Shuler, Michael L.

2006-02-01

A portable fluorescence cytometric system that provides a modular platform for quantitative real-time image measurements has been used to explore the applicability to investigating cellular events on multiple time scales. For a short time scale, we investigated the real-time dynamics of uptake of daunorubicin, a chemotherapeutic agent, in cultured mouse L-cells in a micro cell culture analog compartment using the fluorescent cytometric system. The green fluorescent protein (GFP) expression to monitor induction of pre-specified genes, which occurs on a much longer time scale, has also been measured. Here GFP fluorescence from a doxycycline inducible promoter in a mouse L-cell line was determined. Additionally, a system based on inexpensive LEDs showed performance comparable to a broadband light source based system and reduced photobleaching compared to microscopic examination.

Physical Model of the Dynamic Instability in an Expanding Cell Culture

PubMed Central

Mark, Shirley; Shlomovitz, Roie; Gov, Nir S.; Poujade, Mathieu; Grasland-Mongrain, Erwan; Silberzan, Pascal

2010-01-01

Abstract Collective cell migration is of great significance in many biological processes. The goal of this work is to give a physical model for the dynamics of cell migration during the wound healing response. Experiments demonstrate that an initially uniform cell-culture monolayer expands in a nonuniform manner, developing fingerlike shapes. These fingerlike shapes of the cell culture front are composed of columns of cells that move collectively. We propose a physical model to explain this phenomenon, based on the notion of dynamic instability. In this model, we treat the first layers of cells at the front of the moving cell culture as a continuous one-dimensional membrane (contour), with the usual elasticity of a membrane: curvature and surface-tension. This membrane is active, due to the forces of cellular motility of the cells, and we propose that this motility is related to the local curvature of the culture interface; larger convex curvature correlates with a stronger cellular motility force. This shape-force relation gives rise to a dynamic instability, which we then compare to the patterns observed in the wound healing experiments. PMID:20141748

Separation of somatic and germ cells is required to establish primate spermatogonial cultures.

PubMed

Langenstroth, Daniel; Kossack, Nina; Westernströer, Birgit; Wistuba, Joachim; Behr, Rüdiger; Gromoll, Jörg; Schlatt, Stefan

2014-09-01

Can primate spermatogonial cultures be optimized by application of separation steps and well defined culture conditions? We identified the cell fraction which provides the best source for primate spermatogonia when prolonged culture is desired. Man and marmoset show similar characteristics in regard to germ cell development and function. Several protocols for isolation and culture of human testis-derived germline stem cells have been described. Subsequent analysis revealed doubts on the germline origin of these cells and characterized them as mesenchymal stem cells or fibroblasts. Studies using marmosets as preclinical model confirmed that the published isolation protocols did not lead to propagation of germline cells. Testicular cells derived from nine adult marmoset monkeys (Callithrix jacchus) were cultured for 1, 3, 6 and 11 days and consecutively analyzed for the presence of spermatogonia, differentiating germ cells and testicular somatic cells. Testicular tissue of nine adult marmoset monkeys was enzymatically dissociated and subjected to two different cell culture approaches. In the first approach all cells were kept in the same dish (non-separate culture, n = 5). In the second approach the supernatant cells were transferred into a new dish 24 h after seeding and subsequently supernatant and attached cells were cultured separately (separate culture, n = 4). Real-time quantitative PCR and immunofluorescence were used to analyze the expression of reliable germ cell and somatic markers throughout the culture period. Germ cell transplantation assays and subsequent wholemount analyses were performed to functionally evaluate the colonization of spermatogonial cells. This is the first report revealing an efficient isolation and culture of putative marmoset spermatogonial stem cells with colonization ability. Our results indicate that a separation of spermatogonia from testicular somatic cells is a crucial step during cell preparation. We identified the overgrowth of more rapidly expanding somatic cells to be a major problem when establishing spermatogonial cultures. Initiating germ cell cultures from the supernatant and maintaining germ cells in suspension cultures minimized the somatic cell contamination and provided enriched germ cell fractions which displayed after 11 days of culture a significantly higher expression of germ cell markers genes (DDX-4, MAGE A-4; P < 0.05) compared with separately cultured attached cells. Additionally, germ cell transplantation experiments demonstrated a significantly higher absolute number of cells with colonization ability (P < 0.001) in supernatant cells after 11 days of separate culture. This study presents a relevant aspect for the successful setup of spermatogonial cultures but provides limited data regarding the question of whether the long-term maintenance of spermatogonia can be achieved. Transfer of these preclinical data to man may require modifications of the protocol. Spermatogonial cultures from rodents have become important and innovative tools for basic and applied research in reproductive biology and veterinary medicine. It is expected that spermatogonia-based strategies will be transformed into clinical applications for the treatment of male infertility. Our data in the marmoset monkey may be highly relevant to establish spermatogonial cultures of human testes. Funding was provided by the DFG-Research Unit FOR 1041 Germ Cell Potential (SCHL394/11-2) and by the Graduate Program Cell Dynamics and Disease (CEDAD) together with the International Max Planck Research School - Molecular Biomedicine (IMPRS-MBM). The authors declare that there is no conflict of interest. Not applicable. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bioreactor Expansion of Skin-Derived Precursor Schwann Cells.

PubMed

Walsh, Tylor; Biernaskie, Jeff; Midha, Rajiv; Kallos, Michael S

2016-01-01

Scaling up the production of cells in a culture process is a critical step when trying to develop cell-based regenerative therapies. Static cultures often cannot be easily scaled up to clinically relevant cell numbers. Alternatively, bioreactors offer a highly valuable means to develop a clinical-ready process. To culture adherent cells in suspension, such as skin-derived precursor Schwann cells (SKP-SCs), microcarriers need to be used. Microcarriers are small spherical beads suspended within the vessel that allow for higher growth surface area to volume ratio. Here we describe the procedure of combining microcarriers with the controllability of bioreactors to generate higher cell densities in smaller reactor volumes leading to a more efficient and cost-effective cell production for applications in regenerative medicine.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.

PubMed

Schweizer, Patrick A; Darche, Fabrice F; Ullrich, Nina D; Geschwill, Pascal; Greber, Boris; Rivinius, Rasmus; Seyler, Claudia; Müller-Decker, Karin; Draguhn, Andreas; Utikal, Jochen; Koenen, Michael; Katus, Hugo A; Thomas, Dierk

2017-10-16

Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca 2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

Lentiviral transduction of rat Sertoli cells as a means to modify gene expression

PubMed Central

Nicholls, Peter K.; Stanton, Peter G.; Rainczuk, Katarzyna E.; Qian, Hongwei; Gregorevic, Paul; Harrison, Craig A.

2012-01-01

Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications. PMID:23248769

3D lens-free time-lapse microscopy for 3D cell culture

NASA Astrophysics Data System (ADS)

Berdeu, Anthony; Momey, Fabien; Laperrousaz, Bastien; Bordy, Thomas; Gidrol, Xavier; Dinten, Jean-Marc; Picollet-D'hahan, Nathalie; Allier, Cédric

2017-07-01

We propose a new imaging platform based on lens-free time-lapse microscopy for 3D cell culture and its dedicated algorithm lying on a fully 3D regularized inverse problem approach. First 3D+t results are presented

In vitro 3D regeneration-like growth of human patient brain tissue.

PubMed

Tang-Schomer, M D; Wu, W B; Kaplan, D L; Bookland, M J

2018-05-01

In vitro culture of primary neurons is widely adapted with embryonic but not mature brain tissue. Here, we extended a previously developed bioengineered three-dimensional (3D) embryonic brain tissue model to resected normal patient brain tissue in an attempt to regenerate human neurons in vitro. Single cells and small sized (diameter < 100 μm) spheroids from dissociated brain tissue were seeded into 3D silk fibroin-based scaffolds, with or without collagen or Matrigel, and compared with two-dimensional cultures and scaffold-free suspension cultures. Changes of cell phenotypes (neuronal, astroglial, neural progenitor, and neuroepithelial) were quantified with flow cytometry and analyzed with a new method of statistical analysis specifically designed for percentage comparison. Compared with a complete lack of viable cells in conventional neuronal cell culture condition, supplements of vascular endothelial growth factor-containing pro-endothelial cell condition led to regenerative growth of neurons and astroglial cells from "normal" human brain tissue of epilepsy surgical patients. This process involved delayed expansion of Nestin+ neural progenitor cells, emergence of TUJ1+ immature neurons, and Vimentin+ neuroepithelium-like cell sheet formation in prolonged cultures (14 weeks). Micro-tissue spheroids, but not single cells, supported the brain tissue growth, suggesting importance of preserving native cell-cell interactions. The presence of 3D scaffold, but not hydrogel, allowed for Vimentin+ cell expansion, indicating a different growth mechanism than pluripotent cell-based brain organoid formation. The slow and delayed process implied an origin of quiescent neural precursors in the neocortex tissue. Further optimization of the 3D tissue model with primary human brain cells could provide personalized brain disease models. Copyright © 2018 John Wiley & Sons, Ltd.

Compartmental hollow fiber capillary membrane-based bioreactor technology for in vitro studies on red blood cell lineage direction of hematopoietic stem cells.

PubMed

Housler, Greggory J; Miki, Toshio; Schmelzer, Eva; Pekor, Christopher; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Abbot, Stewart; Zeilinger, Katrin; Gerlach, Jörg C

2012-02-01

Continuous production of red blood cells (RBCs) in an automated closed culture system using hematopoietic stem cell (HSC) progenitor cell populations is of interest for clinical application because of the high demand for blood transfusions. Previously, we introduced a four-compartment bioreactor that consisted of two bundles of hollow fiber microfiltration membranes for transport of culture medium (forming two medium compartments), interwoven with one bundle of hollow fiber membranes for transport of oxygen (O(2)), carbon dioxide (CO(2)), and other gases (forming one gas compartment). Small-scale prototypes were developed of the three-dimensional (3D) perfusion cell culture systems, which enable convection-based mass transfer and integral oxygenation in the cell compartment. CD34(+) HSC were isolated from human cord blood units using a magnetic separation procedure. Cells were inoculated into 2- or 8-mL scaled-down versions of the previously designed 800-mL cell compartment devices and perfused with erythrocyte proliferation and differentiation medium. First, using the small-scale 2-mL analytical scale bioreactor, with an initial seeding density of 800,000 cells/mL, we demonstrated approximately 100-fold cell expansion and differentiation after 7 days of culture. An 8-mL laboratory-scale bioreactor was then used to show pseudocontinuous production by intermediately harvesting cells. Subsequently, we were able to use a model to demonstrate semicontinuous production with up to 14,288-fold expansion using seeding densities of 800,000 cells/mL. The down-scaled culture technology allows for expansion of CD34(+) cells and stimulating these progenitors towards RBC lineage, expressing approximately 40% CD235(+) and enucleation. The 3D perfusion technology provides an innovative tool for studies on RBC production, which is scalable.

Compartmental Hollow Fiber Capillary Membrane–Based Bioreactor Technology for In Vitro Studies on Red Blood Cell Lineage Direction of Hematopoietic Stem Cells

PubMed Central

Housler, Greggory J.; Miki, Toshio; Schmelzer, Eva; Pekor, Christopher; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Abbot, Stewart; Zeilinger, Katrin

2012-01-01

Continuous production of red blood cells (RBCs) in an automated closed culture system using hematopoietic stem cell (HSC) progenitor cell populations is of interest for clinical application because of the high demand for blood transfusions. Previously, we introduced a four-compartment bioreactor that consisted of two bundles of hollow fiber microfiltration membranes for transport of culture medium (forming two medium compartments), interwoven with one bundle of hollow fiber membranes for transport of oxygen (O2), carbon dioxide (CO2), and other gases (forming one gas compartment). Small-scale prototypes were developed of the three-dimensional (3D) perfusion cell culture systems, which enable convection-based mass transfer and integral oxygenation in the cell compartment. CD34+ HSC were isolated from human cord blood units using a magnetic separation procedure. Cells were inoculated into 2- or 8-mL scaled-down versions of the previously designed 800-mL cell compartment devices and perfused with erythrocyte proliferation and differentiation medium. First, using the small-scale 2-mL analytical scale bioreactor, with an initial seeding density of 800,000 cells/mL, we demonstrated approximately 100-fold cell expansion and differentiation after 7 days of culture. An 8-mL laboratory-scale bioreactor was then used to show pseudocontinuous production by intermediately harvesting cells. Subsequently, we were able to use a model to demonstrate semicontinuous production with up to 14,288-fold expansion using seeding densities of 800,000 cells/mL. The down-scaled culture technology allows for expansion of CD34+ cells and stimulating these progenitors towards RBC lineage, expressing approximately 40% CD235+ and enucleation. The 3D perfusion technology provides an innovative tool for studies on RBC production, which is scalable. PMID:21933020

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo.

PubMed

Yang, Yuanheng; Lin, Hang; Shen, He; Wang, Bing; Lei, Guanghua; Tuan, Rocky S

2018-03-15

Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity

NASA Astrophysics Data System (ADS)

Bradley, Jillian H.; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P.; Gregg, Randal K.

2017-11-01

Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When activation of SMG-T cells occurred in SMG, the T cells produced less IL-2 than control T cell cultures upon incubation with PMA and ionomycin. Short-term (24 h) SMG culture and activation of T cells by DC resulted in enhanced IL-2 production compared to Static-T cells, however, when culture was extended to 120 h, SMG-T cells secreted significantly less IL-2 than Static-T cells. SMG-T cell IL-2 doubled upon stimulation of the DC prior to addition to the T cell culture but remained less than control. SMG-T cell resistance to activation appeared comparable to the phenomenon of T cell exhaustion observed in patients with chronic diseases or persistent tumors. That is, long-term culture of T cells in SMG resulted in increased expression of the inhibitory receptor, CTLA-4. Blockade of CTLA-4 interaction with DC ligands resulted in improved T cell IL-2 production. Overall, this is the first study to determine the efficacy of DC in activating peptide-specific T cells. Furthermore, the findings suggests that countermeasures to restore T cell responsiveness in astronauts during long-term spaceflight or those living in microgravity environments should target possible inhibitory pathways that arise on activated T cells following stimulation.

T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity.

PubMed

Bradley, Jillian H; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P; Gregg, Randal K

2017-11-01

Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When activation of SMG-T cells occurred in SMG, the T cells produced less IL-2 than control T cell cultures upon incubation with PMA and ionomycin. Short-term (24 h) SMG culture and activation of T cells by DC resulted in enhanced IL-2 production compared to Static-T cells, however, when culture was extended to 120 h, SMG-T cells secreted significantly less IL-2 than Static-T cells. SMG-T cell IL-2 doubled upon stimulation of the DC prior to addition to the T cell culture but remained less than control. SMG-T cell resistance to activation appeared comparable to the phenomenon of T cell exhaustion observed in patients with chronic diseases or persistent tumors. That is, long-term culture of T cells in SMG resulted in increased expression of the inhibitory receptor, CTLA-4. Blockade of CTLA-4 interaction with DC ligands resulted in improved T cell IL-2 production. Overall, this is the first study to determine the efficacy of DC in activating peptide-specific T cells. Furthermore, the findings suggests that countermeasures to restore T cell responsiveness in astronauts during long-term spaceflight or those living in microgravity environments should target possible inhibitory pathways that arise on activated T cells following stimulation. Copyright © 2017 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

Fetal Fibroblasts and Keratinocytes with Immunosuppressive Properties for Allogeneic Cell-Based Wound Therapy

PubMed Central

Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

2013-01-01

Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors. PMID:23894651

Assessment of genetic and epigenetic variation during long-term Taxus cell culture.

PubMed

Fu, Chunhua; Li, Liqin; Wu, Wenjuan; Li, Maoteng; Yu, Xiaoqing; Yu, Longjiang

2012-07-01

Gradual loss of secondary metabolite production is a common obstacle in the development of a large-scale plant cell production system. In this study, cell morphology, paclitaxel (Taxol®) biosynthetic ability, and genetic and epigenetic variations in the long-term culture of Taxus media cv Hicksii cells were assessed over a 5-year period to evaluate the mechanisms of the loss of secondary metabolites biosynthesis capacity in Taxus cell. The results revealed that morphological variations, gradual loss of paclitaxel yield and decreased transcriptional level of paclitaxel biosynthesis key genes occurred during long-term subculture. Genetic and epigenetic variations in these cultures were also studied at different times during culture using amplified fragment-length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP), and high-performance liquid chromatography (HPLC) analyses. A total of 32 primer combinations were used in AFLP amplification, and none of the AFLP loci were found to be polymorphic, thus no major genetic rearrangements were detected in any of the tested samples. However, results from both MSAP and HPLC indicated that there was a higher level of DNA methylation in the low-paclitaxel yielding cell line after long-term culture. Based on these results, we proposed that accumulation of paclitaxel in Taxus cell cultures might be regulated by DNA methylation. To our knowledge, this is the first report of increased methylation with the prolongation of culture time in Taxus cell culture. It provides substantial clues for exploring the gradual loss of the taxol biosynthesis capacity of Taxus cell lines during long-term subculture. DNA methylation maybe involved in the regulation of paclitaxel biosynthesis in Taxus cell culture.

Establishment and characterization of Xenopus oviduct cells in primary culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marsh, J.; Tata, J.R.

1987-11-01

Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the samemore » characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.« less

Ultra-Soft PDMS-Based Magnetoactive Elastomers as Dynamic Cell Culture Substrata

PubMed Central

Mayer, Matthias; Rabindranath, Raman; Börner, Juliane; Hörner, Eva; Bentz, Alexander; Salgado, Josefina; Han, Hong; Böse, Holger; Probst, Jörn; Shamonin, Mikhail; Monkman, Gareth J.; Schlunck, Günther

2013-01-01

Mechanical cues such as extracellular matrix stiffness and movement have a major impact on cell differentiation and function. To replicate these biological features in vitro, soft substrata with tunable elasticity and the possibility for controlled surface translocation are desirable. Here we report on the use of ultra-soft (Young’s modulus <100 kPa) PDMS-based magnetoactive elastomers (MAE) as suitable cell culture substrata. Soft non-viscous PDMS (<18 kPa) is produced using a modified extended crosslinker. MAEs are generated by embedding magnetic microparticles into a soft PDMS matrix. Both substrata yield an elasticity-dependent (14 vs. 100 kPa) modulation of α-smooth muscle actin expression in primary human fibroblasts. To allow for static or dynamic control of MAE material properties, we devise low magnetic field (≈40 mT) stimulation systems compatible with cell-culture environments. Magnetic field-instigated stiffening (14 to 200 kPa) of soft MAE enhances the spreading of primary human fibroblasts and decreases PAX-7 transcription in human mesenchymal stem cells. Pulsatile MAE movements are generated using oscillating magnetic fields and are well tolerated by adherent human fibroblasts. This MAE system provides spatial and temporal control of substratum material characteristics and permits novel designs when used as dynamic cell culture substrata or cell culture-coated actuator in tissue engineering applications or biomedical devices. PMID:24204603

Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

PubMed

Davari, Maliheh; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

2013-01-01

Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.

Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures

PubMed Central

Davari, Maliheh; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

2013-01-01

Purpose Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. Methods RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2–7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid–binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Results Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum–treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. Conclusions This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases. PMID:24265548

Modelling the tumour microenvironment in long-term microencapsulated 3D co-cultures recapitulates phenotypic features of disease progression.

PubMed

Estrada, Marta F; Rebelo, Sofia P; Davies, Emma J; Pinto, Marta T; Pereira, Hugo; Santo, Vítor E; Smalley, Matthew J; Barry, Simon T; Gualda, Emilio J; Alves, Paula M; Anderson, Elizabeth; Brito, Catarina

2016-02-01

3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of pro-inflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cell-cell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the mono-cultures of MCF-7 cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Microfluidic on-chip biomimicry for 3D cell culture: a fit-for-purpose investigation from the end user standpoint

PubMed Central

Liu, Ye; Gill, Elisabeth; Shery Huang, Yan Yan

2017-01-01

A plethora of 3D and microfluidics-based culture models have been demonstrated in the recent years with the ultimate aim to facilitate predictive in vitro models for pharmaceutical development. This article summarizes to date the progress in the microfluidics-based tissue culture models, including organ-on-a-chip and vasculature-on-a-chip. Specific focus is placed on addressing the question of what kinds of 3D culture and system complexities are deemed desirable by the biological and biomedical community. This question is addressed through analysis of a research survey to evaluate the potential use of microfluidic cell culture models among the end users. Our results showed a willingness to adopt 3D culture technology among biomedical researchers, although a significant gap still exists between the desired systems and existing 3D culture options. With these results, key challenges and future directions are highlighted. PMID:28670465

Microfluidic on-chip biomimicry for 3D cell culture: a fit-for-purpose investigation from the end user standpoint.

PubMed

Liu, Ye; Gill, Elisabeth; Shery Huang, Yan Yan

2017-06-01

A plethora of 3D and microfluidics-based culture models have been demonstrated in the recent years with the ultimate aim to facilitate predictive in vitro models for pharmaceutical development. This article summarizes to date the progress in the microfluidics-based tissue culture models, including organ-on-a-chip and vasculature-on-a-chip. Specific focus is placed on addressing the question of what kinds of 3D culture and system complexities are deemed desirable by the biological and biomedical community. This question is addressed through analysis of a research survey to evaluate the potential use of microfluidic cell culture models among the end users. Our results showed a willingness to adopt 3D culture technology among biomedical researchers, although a significant gap still exists between the desired systems and existing 3D culture options. With these results, key challenges and future directions are highlighted.

Silk screen based dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells.

PubMed

Kamthan, Shweta; Gomes, James; Roychoudhury, Pradip K

2014-08-01

Spin-filters have been primarily used for producing therapeutic proteins from mammalian cells. However, disposability and/or high filter clogging of the existing spin-filter systems affect the process economy and productivity. Hence, to address these drawbacks a reusable dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells was designed. Two non-woven Bombyx mori silk layers were used as filter screen; the outer layer was conducive to cell attachment whilst the inner was non-conducive. Adherent cells can be cultured either in suspended mode using its inner single module or as monolayer of cells using its dual concentric module. We achieved 30 % higher urokinase productivity as compared to the stainless-steel spin-filter during perfusion experiments of adherent human kidney cells in suspended mode. This was due to the hydrophobic and negatively-charged silk screen that allows clog-free perfusion culture for prolonged periods.

Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering

PubMed Central

Wise, Joel K.; Alford, Andrea I.; Goldstein, Steven A.

2014-01-01

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25×106 cells/mL, containing an estimated 5×104 MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2×105 cells/mL) were added to a 65–35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering. PMID:23879621

Comparison of uncultured marrow mononuclear cells and culture-expanded mesenchymal stem cells in 3D collagen-chitosan microbeads for orthopedic tissue engineering.

PubMed

Wise, Joel K; Alford, Andrea I; Goldstein, Steven A; Stegemann, Jan P

2014-01-01

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25 × 10(6) cells/mL, containing an estimated 5 × 10(4) MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2 × 10(5) cells/mL) were added to a 65-35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering.

Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation.

PubMed

Martinez Sanchez, Adela H; Feyerabend, Frank; Laipple, Daniel; Willumeit-Römer, Regine; Weinberg, Annelie; Luthringer, Bérengère J C

2017-03-01

Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10wt% of gadolinium (Mg-10Gd) and Mg with 2wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body. Copyright © 2016 Elsevier B.V. All rights reserved.

Hepatic differentiation of human iPSCs in different 3D models: A comparative study.

PubMed

Meier, Florian; Freyer, Nora; Brzeszczynska, Joanna; Knöspel, Fanny; Armstrong, Lyle; Lako, Majlinda; Greuel, Selina; Damm, Georg; Ludwig-Schwellinger, Eva; Deschl, Ulrich; Ross, James A; Beilmann, Mario; Zeilinger, Katrin

2017-12-01

Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures.

Hepatic differentiation of human iPSCs in different 3D models: A comparative study

PubMed Central

Brzeszczynska, Joanna; Knöspel, Fanny; Armstrong, Lyle; Lako, Majlinda; Greuel, Selina; Damm, Georg; Ludwig-Schwellinger, Eva; Deschl, Ulrich; Ross, James A.

2017-01-01

Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures. PMID:29039463

A microfluidic-based lid device for conventional cell culture dishes to automatically control oxygen level.

PubMed

Lee, Seung Yeob; Yang, Sung

2018-04-25

Most conventional hypoxic cell culture systems undergo reoxygenation during experimental manipulations, resulting in undesirable effects including the reduction of cell viability. A lid device was developed herein for conventional cell culture dishes to resolve this limitation. The integration of multilayered microfluidic channels inside a thin membrane was designed to prevent the reoxygenation caused by reagent infusion and automatically control the oxygen level. The experimental data clearly show the reducibility of the dissolved oxygen in the infusing reagent and the controllability of the oxygen level inside the dish. The feasibility of the device for hypoxia studies was confirmed by HIF-1α experiments. Therefore, the device could be used as a compact and convenient hypoxic cell culture system to prevent reoxygenation-related issues.

Lensless imaging system to quantify cell proliferation

NASA Astrophysics Data System (ADS)

Vinjimore Kesavan, S.; Allier, C. P.; Navarro, F.; Mittler, F.; Chalmond, B.; Dinten, J.-M.

2013-02-01

Owing to its simplicity, lensless imaging system is adept at continuous monitoring of adherent cells inside the incubator. The setup consists of a CMOS sensor with pixel pitch of 2.2 μm and field of view of 24 mm2, LED with a dominating wavelength of 525 nm, along with a pinhole of 150 μm as the source of illumination. The in-line hologram obtained from cells depends on the degree of cell-substrate adhesion. Drastic difference is observed between the holographic patterns of floating and adherent cells. In addition, the well-established fact of reduction of cell-substrate contact during cell division is observed with our system based on corresponding spontaneous transition in the holographic pattern. Here, we demonstrate that by recognizing this specific holographic pattern, number of cells undergoing mitosis in a cell culture with a population of approximately 5000 cells, can be estimated in real-time. The method is assessed on comparison with Edu-based proliferation assay. The approach is straightforward and it eliminates the use of markers to estimate the proliferation rate of a given cell culture. Unlike most proliferation assays, the cells are not harvested enabling continuous monitoring of cell culture.

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds.

PubMed

Jarrahy, Reza; Huang, Weibiao; Rudkin, George H; Lee, Jane M; Ishida, Kenji; Berry, Micah D; Sukkarieh, Modar; Wu, Benjamin M; Yamaguchi, Dean T; Miller, Timothy A

2005-08-01

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.

Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jingu; Park, Sangkyu; Roh, Sangho, E-mail: sangho@snu.ac.kr

A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. Themore » cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.« less

Detection of cancerous cervical cells using physical adhesion of fluorescent silica particles and centripetal force

PubMed Central

Gaikwad, Ravi M.; Dokukin, Maxim E.; Iyer, K. Swaminathan; Woodworth, Craig D.; Volkov, Dmytro O.; Sokolov, Igor

2012-01-01

Here we describe a non-traditional method to identify cancerous human cervical epithelial cells in a culture dish based on physical interaction between silica beads and cells. It is a simple optical fluorescence-based technique which detects the relative difference in the amount of fluorescent silica beads physically adherent to surfaces of cancerous and normal cervical cells. The method utilizes the centripetal force gradient that occurs in a rotating culture dish. Due to the variation in the balance between adhesion and centripetal forces, cancerous and normal cells demonstrate clearly distinctive distributions of the fluorescent particles adherent to the cell surface over the culture dish. The method demonstrates higher adhesion of silica particles to normal cells compared to cancerous cells. The difference in adhesion was initially observed by atomic force microscopy (AFM). The AFM data were used to design the parameters of the rotational dish experiment. The optical method that we describe is much faster and technically simpler than AFM. This work provides proof of the concept that physical interactions can be used to accurately discriminate normal and cancer cells. PMID:21305062

Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

PubMed

Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

2015-08-01

Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids.

PubMed

Hsiao, Amy Y; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R; Pienta, Kenneth J; Takayama, Shuichi

2012-05-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through Z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. Copyright © 2011 Wiley Periodicals, Inc.

384 Hanging Drop Arrays Give Excellent Z-factors and Allow Versatile Formation of Co-culture Spheroids

PubMed Central

Hsiao, Amy Y.; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R.; Pienta, Kenneth J.; Takayama, Shuichi

2012-01-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. PMID:22161651

Poly-ε-caprolactone scaffold and reduced in vitro cell culture: beneficial effect on compaction and improved valvular tissue formation.

PubMed

Brugmans, Marieke M C P; Driessen-Mol, Anita; Rubbens, Mirjam P; Cox, Martijn A J; Baaijens, Frank P T

2015-12-01

Tissue-engineered heart valves (TEHVs), based on polyglycolic acid (PGA) scaffolds coated with poly-4-hydroxybutyrate (P4HB), have shown promising in vivo results in terms of tissue formation. However, a major drawback of these TEHVs is compaction and retraction of the leaflets, causing regurgitation. To overcome this problem, the aim of this study was to investigate: (a) the use of the slowly degrading poly-ε-caprolactone (PCL) scaffold for prolonged mechanical integrity; and (b) the use of lower passage cells for enhanced tissue formation. Passage 3, 5 and 7 (P3, P5 and P7) human and ovine vascular-derived cells were seeded onto both PGA-P4HB and PCL scaffold strips. After 4 weeks of culture, compaction, tissue formation, mechanical properties and cell phenotypes were compared. TEHVs were cultured to observe retraction of the leaflets in the native-like geometry. After culture, tissues based on PGA-P4HB scaffold showed 50-60% compaction, while PCL-based tissues showed compaction of 0-10%. Tissue formation, stiffness and strength were increased with decreasing passage number; however, this did not influence compaction. Ovine PCL-based tissues did render less strong tissues compared to PGA-P4HB-based tissues. No differences in cell phenotype between the scaffold materials, species or cell passage numbers were observed. This study shows that PCL scaffolds may serve as alternative scaffold materials for human TEHVs with minimal compaction and without compromising tissue composition and properties, while further optimization of ovine TEHVs is needed. Reducing cell expansion time will result in faster generation of TEHVs, providing more rapid treatment for patients. Copyright © 2013 John Wiley & Sons, Ltd.

Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

NASA Astrophysics Data System (ADS)

Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

2013-11-01

Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

Sense and sensibility: the use of cell death biomarker assays in high-throughput anticancer drug screening and monitoring treatment responses.

PubMed

Shoshan, Maria C; Havelka, Associate Professor Principal Investigator Aleksandra Mandic; Neumann, Frank; Linder, Stig

2006-11-01

Cell-based screening allows identification of biologically active compounds, for example, potential anticancer drugs. In this review, various screening assays are discussed in terms of what they measure and how this affects interpretation and relevance. High-throughput (HT) assays of viability based on the reduction of exogenous substrates do not always reflect viability or cell number levels. Membrane integrity assays can be used for HT quantification of cell death, but are non-specific as to the death mode. Several HT assays monitor end point apoptosis. Screening libraries at a single concentration (micromolar) can prevent detection of potent apoptosis inducers, as high concentrations may induce mainly necrosis. Using monolayer cultures limits the significance of cell-based screening as the properties of monolayer cells differ from tumours in vivo. Spheroid cultures are more physiological, but are impractical for screening by conventional methods. The authors have developed an assay quantifying accumulation of a caspase-cleaved protein specific for epithelial cells. It provides an integrated measure of apoptosis in two- and three-dimensional cultures and can be used as a blood biomarker assay for tumour apoptosis in vivo.

Cell cultures in drug discovery and development: The need of reliable in vitro-in vivo extrapolation for pharmacodynamics and pharmacokinetics assessment.

PubMed

Jaroch, Karol; Jaroch, Alina; Bojko, Barbara

2018-01-05

For ethical and cost-related reasons, use of animals for the assessment of mode of action, metabolism and/or toxicity of new drug candidates has been increasingly scrutinized in research and industrial applications. Implementation of the 3 "Rs" 1 ; rule (Reduction, Replacement, Refinement) through development of in silico or in vitro assays has become an essential element of risk assessment. Physiologically based pharmacokinetic (PBPK 2 ) modeling is the most potent in silico tool used for extrapolation of pharmacokinetic parameters to animal or human models from results obtained in vitro. Although, many types of in vitro assays are conducted during drug development, use of cell cultures is the most reliable one. Two-dimensional (2D) cell cultures have been a part of drug development for many years. Nowadays, their role is decreasing in favor of three-dimensional (3D) cell cultures and co-cultures. 3D cultures exhibit protein expression patterns and intercellular junctions that are closer to in vivo states in comparison to classical monolayer cultures. Co-cultures allow for examinations of the mutual influence of different cell lines. However, the complexity and high costs of co-cultures and 3D equipment exclude such methods from high-throughput screening (HTS). 3 In vitro absorption, distribution, metabolism, and excretion assessment, as well as drug-drug interaction (DDI), are usually performed with the use of various cell culture based assays. Progress in in silico and in vitro methods can lead to better in vitro-in vivo extrapolation (IVIVE 4 ) outcomes and have a potential to contribute towards a significant reduction in the number of laboratory animals needed for drug research. As such, concentrated efforts need to be spent towards the development of an HTS in vitro platform with satisfactory IVIVE features. Copyright © 2017 Elsevier B.V. All rights reserved.

Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

PubMed

de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

2010-12-15

Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

PubMed

Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S

2016-10-01

3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. © 2016 Society for Laboratory Automation and Screening.

Shear-wave elasticity measurements of three-dimensional cell cultures for mechanobiology

PubMed Central

Kuo, Po-Ling; Charng, Ching-Che; Wu, Po-Chen

2017-01-01

ABSTRACT Studying mechanobiology in three-dimensional (3D) cell cultures better recapitulates cell behaviors in response to various types of mechanical stimuli in vivo. Stiffening of the extracellular matrix resulting from cell remodeling potentiates many pathological conditions, including advanced cancers. However, an effective tool for measuring the spatiotemporal changes in elastic properties of such 3D cell cultures without directly contacting the samples has not been reported previously. We describe an ultrasonic shear-wave-based platform for quantitatively evaluating the spatiotemporal dynamics of the elasticity of a matrix remodeled by cells cultured in 3D environments. We used this approach to measure the elasticity changes of 3D matrices grown with highly invasive lung cancer cells and cardiac myoblasts, and to delineate the principal mechanism underlying the stiffening of matrices remodeled by these cells. The described approach can be a useful tool in fields investigating and manipulating the mechanotransduction of cells in 3D contexts, and also has potential as a drug-screening platform. PMID:27505887

[The proliferative characteristics of cells in culture during perfusion of the medium].

PubMed

Akatov, V S; Lavrovskaia, V P; Lezhnev, E I

1991-01-01

The proliferation of Chinese hamster fibroblasts (CHF) and NIH 3T3 cells was studied at different flow rates immediately above the cells. It is shown that there is a limiting density of saturation of the perfused culture that accounts for 1.7 x 10(6) - 2.0 x 10(6) cells/cm2 for NIH 3T3 cells, and for 6 x 10(6) - 7 x 10(6) cells/cm2 for CHF. The growth curves and the distribution of cells with respect to the phases of the cell cycle during cultivation with and without perfusion are presented. Based on the results obtained it is assumed that the limit of saturation density of perfused CHF culture is due to the mass transfer of the growth-inhibiting metabolites inside the multilayer structures. The assumption seems to hold true for NIH 3T3 cells, too, even though the multilayer character of growth of this culture is less pronounced than in CHF.

An evaluation of the inflammatory response of lipopolysaccharide-treated primary dental pulp cells with regard to calcium silicate-based cements

PubMed Central

Lai, Wei-Yun; Kao, Chia-Tze; Hung, Chi-Jr; Huang, Tsui-Hsien; Shie, Ming-You

2014-01-01

This study compared the biological changes of lipopolysaccharide (LPS)-treated dental pulp (DP) cells directly cultured on mineral trioxide aggregate (MTA) and calcium silicate (CS) cements. DP cells were treated with LPS for 24 h. Then, the LPS-treated DP cells were cultured on MTA or CS cements. Cell viability, cell death mechanism and interleukin (IL)-1β expressions were analysed. A one-way analysis of variance was used to evaluate the significance of the differences between the means. A significantly higher IL-1β expression (2.9-fold) was found for LPS-treated cells (P<0.05) compared with DP cells without LPS treatment at 24 h. Absorbance values of LPS-treated cells cultured on CS cement were higher than a tissue culture plate. A significant difference (P<0.05) in cell viability was observed between cells on CS and MTA cements 24 h after seeding. At 48 h, a high concentration of Si (5 mM) was released from MTA, which induced LPS-treated DP cell apoptosis. The present study demonstrates that CS cement is biocompatible with cultured LPS-treated DP cells. MTA stimulates inflammation in LPS-treated DP cells, which leads to greater IL-1β expression and apoptosis. PMID:24556955

Cryopreservation Method for the Effective Collection of Dental Pulp Stem Cells.

PubMed

Takebe, Yusuke; Tatehara, Seiko; Fukushima, Tatsuhiro; Tokuyama-Toda, Reiko; Yasuhara, Rika; Mishima, Kenji; Satomura, Kazuhito

2017-05-01

Dental pulp stem cells (DPSCs) are an attractive cell source for use in cell-based therapy, regenerative medicine, and tissue engineering because DPSCs have a high cell proliferation ability and multidifferentiation capacity. However, several problems are associated with the collection and preservation of DPSCs for use in future cell-based therapy. In particular, the isolation of DPSCs for cryopreservation is time consuming and expensive. In this study, we developed a novel cryopreservation method (NCM) for dental pulp tissues to isolate suitable DPSCs after thawing cryopreserved tissue. Using the NCM, dental pulp tissues were cultured on adhesion culture dishes for 5 days and then cryopreserved. After thawing, the cryopreserved dental pulp tissue fragments exhibited cell migration. We evaluated each property of DPSCs isolated using the NCM (DPSCs-NCM) and the explant method alone without cryopreservation (DPSCs-C). DPSCs-NCM had the same proliferation capacity as DPSCs-C. Flow cytometry (FACS) analysis indicated that both DPSCs-NCM and DPSCs-C were positive for mesenchymal stem cell markers at the same level but negative for hematopoietic cell markers. Moreover, both DPSCs-NCM and DPSCs-C could differentiate into osteogenic, chondrogenic, and adipogenic cells during culture in each induction medium. These results suggest that DPSCs-NCM may be mesenchymal stem cells. Therefore, our novel method might facilitate the less expensive cryopreservation of DPSCs, thereby providing suitable DPSCs for use in patients in future cell-based therapies.

Centrifugation assay for measuring adhesion of serially passaged bovine chondrocytes to polystyrene surfaces.

PubMed

Kaplan, David S; Hitchins, Victoria M; Vegella, Thomas J; Malinauskas, Richard A; Ferlin, Kimberly M; Fisher, John P; Frondoza, Carmelita G

2012-07-01

A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.

Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

PubMed

Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

2010-12-01

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Manufacturing of dental pulp cell-based products from human third molars: current strategies and future investigations

PubMed Central

Ducret, Maxime; Fabre, Hugo; Degoul, Olivier; Atzeni, Gianluigi; McGuckin, Colin; Forraz, Nico; Alliot-Licht, Brigitte; Mallein-Gerin, Frédéric; Perrier-Groult, Emeline; Farges, Jean-Christophe

2015-01-01

In recent years, mesenchymal cell-based products have been developed to improve surgical therapies aimed at repairing human tissues. In this context, the tooth has recently emerged as a valuable source of stem/progenitor cells for regenerating orofacial tissues, with easy access to pulp tissue and high differentiation potential of dental pulp mesenchymal cells. International guidelines now recommend the use of standardized procedures for cell isolation, storage and expansion in culture to ensure optimal reproducibility, efficacy and safety when cells are used for clinical application. However, most dental pulp cell-based medicinal products manufacturing procedures may not be fully satisfactory since they could alter the cells biological properties and the quality of derived products. Cell isolation, enrichment and cryopreservation procedures combined to long-term expansion in culture media containing xeno- and allogeneic components are known to affect cell phenotype, viability, proliferation and differentiation capacities. This article focuses on current manufacturing strategies of dental pulp cell-based medicinal products and proposes a new protocol to improve efficiency, reproducibility and safety of these strategies. PMID:26300779

Maintenance of human adipose derived stem cell (hASC) differentiation capabilities using a 3D culture.

PubMed

Lin, Ching-Yu; Huang, Chi-Hui; Wu, Yuan-Kun; Cheng, Nai-Chen; Yu, Jiashing

2014-07-01

In this study, 3D culture system for human adipose-derived stem cell (hASC) using a BioLevitator as the bioreactor for microcarrier-based cultures was established. During the culturing period, hASCs preferred to grow in crevices between microcarriers and a high viability was maintained even when reaching confluency. Adipogenic or osteogenic differential medium was used to induce hASCs and differential potentials of these cells were compared between 2D and 3D environments via RT-PCR and staining quantifications. CEBP/α gene expression was significant higher in 3D condition at day 21 (P < 0.05). Staining quantification indicates that cells cultured in 3D condition have significant better differentiation potential from day 14 to 21 for both adipogenic and osteogenic lineages (P < 0.01).

Extracellular matrix components direct porcine muscle stem cell behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

2010-02-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatinmore » and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.« less

Cell motion predicts human epidermal stemness

PubMed Central

Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

2015-01-01

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083

Three-Dimensional Cell Culture Models for Infectious Disease and Drug Development

NASA Technical Reports Server (NTRS)

Nickerson, Cheryl A.; Honer zu Bentrup, Kerstin; Ott, C. Mark

2005-01-01

Three-dimensional (3-D) cell cultures hold enormous potential to advance our understanding of infectious disease and to effectively translate basic cellular research into clinical applications. Using novel NASA bioreactor technology, the rotating wall vessel (RWV), we have engineered physiologically relevant 3-D human tissue culture models for infectious disease studies. The design of the RWV is based on the understanding that organs and tissues function in a 3-D environment, and that this 3-D architecture is critical for the differentiated form and function of tissues in vivo. The RWV provides large numbers of cells which are amenable to a wide variety of experimental manipulations and provides an easy, reproducible, and cost-effective approach to enhance differentiated features of cell culture models.

Induction of Three-Dimensional Growth of Human Liver Cells in Simulated Microgravity

NASA Technical Reports Server (NTRS)

Pellis, Neal R.; Khaoustov, V. I.; Yoffe, B.; Murry, D. J.; Soriano, H. E.; Risin, D.; Dawson, David L. (Technical Monitor)

1999-01-01

We previously reported that a NASA-developed bioreactor that simulates microgravity environment and creates the unique environment of low shear force and high-mass transfer is conducive for maintaining long term 3-D cell cultures of functional hepatocytes (60 days). However, significant further expansion of liver mass, or the remodeling of liver in vitro was jeopardized by the appearance of apoptotic zones in the center of large cell aggregates. To optimize oxygenation and nutritional uptake within growing cellular aggregates we cultured primary human liver cells (HLC) in a bioreactor in the presence or absence of microcarriers and biodegradable scaffolds. Also, to promote angiogenesis, HLC were cultured with or without microvascular endothelial cells. HLC were harvested from human livers by collagenase perfusion. While microcarriers did not affect cell growth, HLC cultured with biodegradable scaffolds made from polyglycolic acid (PGA) formed aggregates up to 3 cm in length. Culturing cells with PGA scaffolds increased the efficiency of cell self-assembly and the formation of larger cell aggregates. Based on histological evaluation it appears that the degree of apoptotic cells was diminished as compared to cultures without scaffolds. Histology of HLC with PGA-scaffolds revealed cell distribution between the fibers of the scaffolds, and cell-cell and cell-fiber interactions. Analyses of HLC spheroids revealed tissue-like structures comprised of hepatocytes, biliary epithelial cells and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes and bile canaliculi with multiple microvilli and tight cellular junctions. Hepatocytes were further organized into tight clusters surrounded by complex stromal structures and reticulin fibers. Also, we observed higher levels of albumin mRNA expression when hepatocytes were co-cultured with endothelial cells. To evaluate viability and microsomal deethylation activity of hepatocytes we assessed the metabolism of midazolam by gas chromatography. Samples that were collected from HLC cultured in the bioreactor contained higher levels of midazolam metabolites (1-OH-midazolam and 4-OH-midazolam) than samples collected from conventional cultures. In summary, we have shown that co-culture of HLC with endothelial cells and/or culturing in the presence of PGA scaffolds provides additional advantages in maintaining functional activity of 3-D hepatocyte cultures that resemble liver tissue. This cell culture system may facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo and suggest the feasibility of using this approach for pre-clinical metabolic screening of novel drug candidates.

Unique differentiation profile of mouse embryonic stem cells in rotary and stirred tank bioreactors.

PubMed

Fridley, Krista M; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B; Roy, Krishnendu

2010-11-01

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.

FGF1 and IGF1-conditioned 3D culture system promoted the amplification and cancer stemness of lung cancer cells.

PubMed

Liu, Pengpeng; Zhang, Rui; Yu, Wenwen; Ye, Yingnan; Cheng, Yanan; Han, Lei; Dong, Li; Chen, Yongzi; Wei, Xiyin; Yu, Jinpu

2017-12-01

Lung cancer stem cells (LCSCs) are considered as the cellular origins of metastasis and relapse of lung cancer. However, routine two-dimensional culture system (2D-culture) hardly mimics the growth and functions of LCSCs in vivo and therefore significantly decreases the stemness activity of LCSCs. In this study, we constructed a special BME-based three-dimensional culture system (3D-culture) to amplify LCSCs in human lung adenocarcinoma cell line A549 cells and found 3D-culture promoted the enrichment and amplification of LCSCs in A549 cells displaying higher proliferation potential and invasion activity, but lower apoptosis. The expression and secretion levels of FGF1 and IGF1 were dramatically elevated in 3D-culture compared to 2D-culture. After growing in FGF1 and IGF1-conditioned 3D-culture, the proportion of LCSCs with specific stemness phenotypes in A549 cells significantly increased compared to that in conventional 3D suspension culture system. Further results indicated that FGF1 and IGF1 promoted the amplification and cancer stemness of LCSCs dependent on MAPK signaling pathway. Our data firstly established a growth factors-conditioned 3D-culture for LCSCs and demonstrated the effects of FGF1 and IGF1 in promoting the enrichment and amplification of LCSCs which might provide a feasible cell model in vitro for both mechanism study and translational research on lung cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Export of an invertase by yeast cells (Candida utilis)].

PubMed

Alekseeva, O V; Sabirzianova, T A; Celiakh, I O; Kalebina, T S; Kulaev, I S

2014-01-01

Export and accumulation of various forms of invertase (EC 3.2.1.26) in the cell wall and culture liquid of the yeast Candida utilis was investigated. It was found that the high-molecular-weight CW-form of invertase is present in the cell wall. This form is not exported into the culture liquid, and it is by a third more glycosylated than the previously described exported S-form. It was shown that one of the two liquid forms of invertase exported into the culture-the glycosylated S-form--is retained in the cell wall, while the other one--the nonglycosylated F-form--was not detected in the cell wall. Based on these results, as well as data on the distribution dynamics of the enzyme in the culture liquid and in the cell wall during different growth stages of a yeast culture, we suggested that the nonglycosylated form was exported into the culture liquid via the zone of abnormal cell wall permeability and the glycosylated forms of this enzyme (both exported and nonexported) did not use this pathway (the degree of N-glycosylation is an important factor determining the final localization of the enzyme).

3D culture of Her2+ breast cancer cells promotes AKT to MAPK switching and a loss of therapeutic response.

PubMed

Gangadhara, Sharath; Smith, Chris; Barrett-Lee, Peter; Hiscox, Stephen

2016-06-01

The Her2 receptor is overexpressed in up to 25 % of breast cancers and is associated with a poor prognosis. Around half of Her2+ breast cancers also express the estrogen receptor and treatment for such tumours can involve both endocrine and Her2-targeted therapies. However, despite preclinical data supporting the effectiveness of these agents, responses can vary widely in the clinical setting. In light of the increasing evidence pointing to the interplay between the tumour and its extracellular microenvironment as a significant determinant of therapeutic sensitivity and response here we investigated the impact of 3D matrix culture of breast cancer cells on their therapeutic sensitivity. A 3D Matrigel-based culture system was established and optimized for the growth of ER+/Her2+ breast cancer cell models. Growth of cells in response to trastuzumab and endocrine agents in 3D culture versus routine monolayer culture were assessed using cell counting and Ki67 staining. Endogenous and trastuzumab-modulated signalling pathway activity in 2D and 3D cultures were assessed using Western blotting. Breast cancer cells in 3D culture displayed an attenuated response to both endocrine agents and trastuzumab compared with cells cultured in traditional 2D monolayers. Underlying this phenomenon was an apparent matrix-induced shift from AKT to MAPK signalling; consequently, suppression of MAPK in 3D cultures restores therapeutic response. These data suggest that breast cancer cells in 3D culture display a reduced sensitivity to therapeutic agents which may be mediated by internal MAPK-mediated signalling. Targeting of adaptive pathways that maintain growth in 3D culture may represent an effective strategy to improve therapeutic response clinically.

Unique cell culture systems for ground based research

NASA Technical Reports Server (NTRS)

Lewis, Marian L.

1990-01-01

The horizontally rotating fluid-filled, membrane oxygenated bioreactors developed at NASA Johnson for spacecraft applications provide a powerful tool for ground-based research. Three-dimensional aggregates formed by cells cultured on microcarrier beads are useful for study of cell-cell interactions and tissue development. By comparing electron micrographs of plant seedlings germinated during Shuttle flight 61-C and in an earth-based rotating bioreactor it is shown that some effects of microgravity are mimicked. Bioreactors used in the UAH Bioreactor Laboratory will make it possible to determine some of the effects of altered gravity at the cellular level. Bioreactors can be valuable for performing critical, preliminary-to-spaceflight experiments as well as medical investigations such as in vitro tumor cell growth and chemotherapeutic drug response; the enrichment of stem cells from bone marrow; and the effect of altered gravity on bone and muscle cell growth and function and immune response depression.

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement.

PubMed

Shieh, Hester F; Graham, Christopher D; Brazzo, Joseph A; Zurakowski, David; Fauza, Dario O

2017-06-01

We sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair. Human afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05). There was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96). Among clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair. N/A (animal and laboratory study). Copyright © 2017 Elsevier Inc. All rights reserved.

Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

PubMed

Roa, Jinae N; Tresguerres, Martin

2016-08-01

Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology. Copyright © 2016 the American Physiological Society.

Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

PubMed

Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

2017-02-28

Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

PubMed

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

PubMed Central

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system. PMID:24465853

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine.

PubMed

Ruiz, Christian; Kustermann, Stefan; Pietilae, Elina; Vlajnic, Tatjana; Baschiera, Betty; Arabi, Leila; Lorber, Thomas; Oeggerli, Martin; Savic, Spasenija; Obermann, Ellen; Singer, Thomas; Rothschild, Sacha I; Zippelius, Alfred; Roth, Adrian B; Bubendorf, Lukas

2016-01-01

The use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs. MPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists. We have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs. We developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.

Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Spaulding, Glenn F.; Tsao, Yow-Min D.; Flechsig, Scott; Jones, Leslie; Soehnge, Holly

2003-01-01

The hydrodynamic focusing bioreactor (HFB) is a bioreactor system designed for three-dimensional cell culture and tissue-engineering investigations on orbiting spacecraft and in laboratories on Earth. The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear culture environment simultaneously with the "herding" of suspended cells, tissue assemblies, and air bubbles. Under development for use in the Biotechnology Facility on the International Space Station, the HFB has successfully grown large three-dimensional, tissuelike assemblies from anchorage-dependent cells and grown suspension hybridoma cells to high densities. The HFB, based on the principle of hydrodynamic focusing, provides the capability to control the movement of air bubbles and removes them from the bioreactor without degrading the low-shear culture environment or the suspended three-dimensional tissue assemblies. The HFB also provides unparalleled control over the locations of cells and tissues within its bioreactor vessel during operation and sampling.

Visual Estimation of Bacterial Growth Level in Microfluidic Culture Systems.

PubMed

Kim, Kyukwang; Kim, Seunggyu; Jeon, Jessie S

2018-02-03

Microfluidic devices are an emerging platform for a variety of experiments involving bacterial cell culture, and has advantages including cost and convenience. One inevitable step during bacterial cell culture is the measurement of cell concentration in the channel. The optical density measurement technique is generally used for bacterial growth estimation, but it is not applicable to microfluidic devices due to the small sample volumes in microfluidics. Alternately, cell counting or colony-forming unit methods may be applied, but these do not work in situ; nor do these methods show measurement results immediately. To this end, we present a new vision-based method to estimate the growth level of the bacteria in microfluidic channels. We use Fast Fourier transform (FFT) to detect the frequency level change of the microscopic image, focusing on the fact that the microscopic image becomes rough as the number of cells in the field of view increases, adding high frequencies to the spectrum of the image. Two types of microfluidic devices are used to culture bacteria in liquid and agar gel medium, and time-lapsed images are captured. The images obtained are analyzed using FFT, resulting in an increase in high-frequency noise proportional to the time passed. Furthermore, we apply the developed method in the microfluidic antibiotics susceptibility test by recognizing the regional concentration change of the bacteria that are cultured in the antibiotics gradient. Finally, a deep learning-based data regression is performed on the data obtained by the proposed vision-based method for robust reporting of data.

Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery.

PubMed

Stock, Kristin; Estrada, Marta F; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-07-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.

Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery

PubMed Central

Stock, Kristin; Estrada, Marta F.; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E.; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C.; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-01-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models. PMID:27364600

Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface

PubMed Central

Casaroli-Marano, Ricardo P.; Nieto-Nicolau, Núria; Martínez-Conesa, Eva M.; Edel, Michael; Álvarez-Palomo, Ana B.

2015-01-01

The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea’s transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement—cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET)—present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD. PMID:26239129

Comparison between Flow Cytometry and Traditional Culture Methods for Efficacy Assessment of Six Disinfectant Agents against Nosocomial Bacterial Species

PubMed Central

Massicotte, Richard; Mafu, Akier A.; Ahmad, Darakhshan; Deshaies, Francis; Pichette, Gilbert; Belhumeur, Pierre

2017-01-01

The present study was undertaken to compare the use of flow cytometry (FCM) and traditional culture methods for efficacy assessment of six disinfectants used in Quebec hospitals including: two quaternary ammonium-based, two activated hydrogen peroxide-based, one phenol-based, and one sodium hypochlorite-based. Four nosocomial bacterial species, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Vancomycin-resistant Enterococci faecalis, were exposed to minimum lethal concentrations (MLCs) and sublethal concentrations (1/2 MLCs) of disinfectants under study. The results showed a strong correlation between the two techniques for the presence of dead and live cell populations, as well as, evidence of injured populations with the FCM. The only exception was observed with sodium hypochlorite at higher concentrations where fluorescence was diminished and underestimating dead cell population. The results also showed that FCM can replace traditional microbiological methods to study disinfectant efficacy on bacteria. Furthermore, FCM profiles for E. coli and E. faecalis cells exposed to sublethal concentrations exhibited distinct populations of injured cells, opening a new aspect for future research and investigation to elucidate the role of injured, cultural/noncuturable/resuscitable cell populations in infection control. PMID:28217115

Cell division in Escherichia coli cultures monitored at single cell resolution

PubMed Central

Roostalu, Johanna; Jõers, Arvi; Luidalepp, Hannes; Kaldalu, Niilo; Tenson, Tanel

2008-01-01

Background A fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate. Results We monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP) upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency. Conclusion In principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular organisms other than E. coli. PMID:18430255

Transfection in perfused microfluidic cell culture devices: A case study.

PubMed

Raimes, William; Rubi, Mathieu; Super, Alexandre; Marques, Marco P C; Veraitch, Farlan; Szita, Nicolas

2017-08-01

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.

Intra-discal injection of autologous, hypoxic cultured bone marrow-derived mesenchymal stem cells in five patients with chronic lower back pain: a long-term safety and feasibility study.

PubMed

Elabd, Christian; Centeno, Christopher J; Schultz, John R; Lutz, Gregory; Ichim, Thomas; Silva, Francisco J

2016-09-01

Chronic low back pain due to disc degeneration represents a major social and economic burden worldwide. The current standard of care is limited to symptomatic relief and no current approved therapy promotes disc regeneration. Bone marrow-derived mesenchymal stem cells (MSCs) are easily accessible and well characterized. These MSCs are multipotent and exhibit great tissue regenerative potential including bone, cartilage, and fibrous tissue regeneration. The use of this cell-based biologic for treating protruding disc herniation and/or intervertebral disc degeneration is a promising therapeutic strategy, due to their known regenerative, immuno-modulatory and anti-inflammatory properties. Five patients diagnosed with degenerative disc disease received an intra-discal injection of autologous, hypoxic cultured, bone marrow-derived mesenchymal stem cells (15.1-51.6 million cells) as part of a previous study. These patients were re-consented to participate in this study in order to assess long-term safety and feasibility of intra-discal injection of autologous, hypoxic cultured, bone marrow-derived mesenchymal stem cells 4-6 years post mesenchymal stem cell infusion. The follow-up study consisted of a physical examination, a low back MRI, and a quality of life questionnaire. Patients' lower back MRI showed absence of neoplasms or abnormalities surrounding the treated region. Based on the physical examination and the quality of life questionnaire, no adverse events were reported due to the procedure or to the stem cell treatment 4-6 years post autologous, hypoxic cultured mesenchymal stem cell infusion. All patients self-reported overall improvement, as well as improvement in strength, post stem cell treatment, and four out of five patients reported improvement in mobility. This early human clinical data suggests the safety and feasibility of the clinical use of hypoxic cultured bone marrow-derived mesenchymal stem cells for the treatment of lower back pain due to degenerative disc disorders and support further studies utilizing hypoxic cultured bone marrow-derived stem cells. The overall improvements reported are encouraging, but a larger double-blind, controlled, randomized clinical study with significant number of patients and implementation of validated endpoint measurements are next steps in order to demonstrate efficacy of this cell-based biologic.

Disarmed by density

PubMed Central

Nasi, Aikaterini; Rethi, Bence

2013-01-01

We observed a cell concentration-dependent differentiation switch among cultured dendritic cells (DCs) triggered by lactic acid, a product of glycolytic metabolism. In particular, while interleukin (IL)-12, IL-23, and tumor necrosis factor α (TNFα)-producing, migratory DCs developed in sparse cultures, IL-10-producing, non-migratory DCs differentiated in dense cultures. This points to a novel opportunity for tailoring DC-based anticancer therapies through metabolism modulation in developing DCs. PMID:24575378

In vitro micro-physiological immune-competent model of the human skin.

PubMed

Ramadan, Qasem; Ting, Fiona Chia Wan

2016-05-21

Skin allergy, in particular, allergic contact dermatitis and irritant contact dermatitis, are common occupational and environmental health problems affecting the quality of life of a significant proportion of the world population. Since all new ingredients to be incorporated into a product are potential skin allergens, it is essential that these ingredients be first tested for their allergenic potential. However, despite the considerable effort using animal models to understand the underlying mechanism of skin sensitization, to date, the molecular and cellular responses due to skin contact with sensitizers are still not fully understood. To replace animal testing and to improve the prediction of skin sensitization, significant attention has been directed to the use of reconstructed organotypic in vitro models of human skin. Here we describe a miniaturized immune competent in vitro model of human skin based on 3D co-culture of immortalized human keratinocytes (HaCaT) as a model of the epidermis barrier and human leukemic monocyte lymphoma cell line (U937) as a model of human dendritic cells. The biological model was fitted in a microfluidic-based cell culture system that provides a dynamic cellular environment that mimics the in vivo environment of skin. The dynamic perfusion of culture media significantly improved the tight junction formation as evidenced by measuring higher values of TEER compared to static culture. This setting also maintained the high viability of cells over extended periods of time up to 17 days. The perfusion-based culture also allows growth of the cells at the air-liquid interface by exposing the apical side of the cells to air while providing the cell nutrients through a basolateral fluidic compartment. The microsystem has been evaluated to investigate the effect of the chemical and physical (UV irradiation) stimulation on the skin barrier (i.e. the TJ integrity). Three-tiered culture differential stimulation allowed the investigation of the role of the keratinocyte layer as a protection barrier to chemical/biological hazards.

Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirano, Kunio; Konagaya, Shuhei; Turner, Alexander

Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregatesmore » were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality. - Highlights: • A simple, closed-channel-based, semi-automatic culture system is proposed. • Uniform cell aggregate formation and culture is realized in microwell structure. • Functional islet cells are successfully induced following 30-plus-day protocol. • System requires no daily medium replacement and reduces contamination risk.« less

Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lam, Alan Tin-Lun, E-mail: alan_lam@bti.a-star.edu.sg; Chen, Allen Kuan-Liang; Ting, Sherwin Qi-Peng

Current methods for human pluripotent stem cells (hPSC) expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are expanded as cell/MC aggregates and then directly differentiated as embryoid bodies (EBs) in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. This review describes the principles ofmore » such microcarriers based integrated hPSC expansion and differentiation process, and parameters that can affect its efficiency (such as MC type and extracellular matrix proteins coatings, cell/MC aggregates size, and agitation). Finally examples of integrated process for generation cardiomyocytes (CM) and neural progenitor cells (NPC) as well as challenges to be solved are described. - Highlights: • Expansion of hPSC on microcarriers. • Differentiation of hPSC on microcarriers. • Parameters that can affect the expansion and differentiation of hPSC on microcarriers. • Integration of expansion and differentiation of hPSC on microcarriers in one unit operation.« less

Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

PubMed

Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

2014-10-01

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

Medical Research System

NASA Technical Reports Server (NTRS)

1993-01-01

Based on Johnson Space Flight Center's development of a rotating bioreactor cell culture apparatus for Space Shuttle medical research, Johnson Space Flight Center engineers who worked on the original project formed a company called Synthecon, with the intention of commercializing the bioreactor technology. Synthecon grows three dimensional tissues in the bioreactor. These are superior to previous two-dimensional tissue samples in the study of human cell growth. A refined version of the Johnson Space Center technology, Synthecon's Rotary Cell Culture System includes a cell culture chamber that rotates around a horizontal axis. The cells establish an orbit that approximates free fall through the liquid medium in the chamber. The technology has significant applications for cancer research and treatment as well as AIDS research.

Metabolic design of macroscopic bioreaction models: application to Chinese hamster ovary cells.

PubMed

Provost, A; Bastin, G; Agathos, S N; Schneider, Y-J

2006-12-01

The aim of this paper is to present a systematic methodology to design macroscopic bioreaction models for cell cultures based upon metabolic networks. The cell culture is seen as a succession of phases. During each phase, a metabolic network represents the set of reactions occurring in the cell. Then, through the use of the elementary flux modes, these metabolic networks are used to derive macroscopic bioreactions linking the extracellular substrates and products. On this basis, as many separate models are obtained as there are phases. Then, a complete model is obtained by smoothly switching from model to model. This is illustrated with batch cultures of Chinese hamster ovary cells.

DEVELOPMENT OF AN INTEGRATED CELL CULTURE/RT-PCR METHOD FOR THE DETECTION OF ENTEROVIRUS IN WATER

EPA Science Inventory

Virus contamination in environmental samples is believed to be underestimated due to the limitations in the methods available for detection. A major detection method is based upon the formation of cytopathic effect (CPE) in cell culture. The main limitations to this method are ...

Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells

PubMed Central

Loureiro, Renata Ruoco; Cristovam, Priscila Cardoso; Martins, Caio Marques; Covre, Joyce Luciana; Sobrinho, Juliana Aparecida; Ricardo, José Reinaldo da Silva; Hazarbassanov, Rossen Myhailov; Höfling-Lima, Ana Luisa; Belfort, Rubens; Nishi, Mauro

2013-01-01

Purpose To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells. Methods Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5′-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT–PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining. Results Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM. Conclusions Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM. PMID:23378720

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

PubMed

Yamazaki, Taisuke; Wakai, Mariko; Enosawa, Shin; Tokiwa, Takayoshi

2017-06-01

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

PubMed

Ayaydin, Ferhan; Kotogány, Edit; Ábrahám, Edit; Horváth, Gábor V

2017-01-01

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

PubMed Central

LAPTEVA, NATALIA; DURETT, APRIL G.; SUN, JIALI; ROLLINS, LISA A.; HUYE, LESLIE L.; FANG, JIAN; DANDEKAR, VARADA; MEI, ZHUYONG; JACKSON, KIMBERLEY; VERA, JUAN; ANDO, JUN; NGO, MINHTRAN C.; COUSTAN-SMITH, ELAINE; CAMPANA, DARIO; SZMANIA, SUSANN; GARG, TARUN; MORENO-BOST, AMBERLY; VANRHEE, FRITS; GEE, ADRIAN P.; ROONEY, CLIONA M.

2016-01-01

Background aims Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results Using this system we produced up to 19 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 CD3− CD56+ NK cells, within 8–10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8+ T cells within the NK cultures. However, these CD3+ T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy. PMID:22900959

Good Cell Culture Practice for stem cells and stem-cell-derived models.

PubMed

Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Yamazaki, Daiju; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas

2017-01-01

The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

Generic Raman-based calibration models enabling real-time monitoring of cell culture bioreactors.

PubMed

Mehdizadeh, Hamidreza; Lauri, David; Karry, Krizia M; Moshgbar, Mojgan; Procopio-Melino, Renee; Drapeau, Denis

2015-01-01

Raman-based multivariate calibration models have been developed for real-time in situ monitoring of multiple process parameters within cell culture bioreactors. Developed models are generic, in the sense that they are applicable to various products, media, and cell lines based on Chinese Hamster Ovarian (CHO) host cells, and are scalable to large pilot and manufacturing scales. Several batches using different CHO-based cell lines and corresponding proprietary media and process conditions have been used to generate calibration datasets, and models have been validated using independent datasets from separate batch runs. All models have been validated to be generic and capable of predicting process parameters with acceptable accuracy. The developed models allow monitoring multiple key bioprocess metabolic variables, and hence can be utilized as an important enabling tool for Quality by Design approaches which are strongly supported by the U.S. Food and Drug Administration. © 2015 American Institute of Chemical Engineers.

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

PubMed Central

Iwamaru, Yoshifumi; Takenouchi, Takato; Imamura, Morikazu; Shimizu, Yoshihisa; Miyazawa, Kohtaro; Mohri, Shirou; Yokoyama, Takashi

2013-01-01

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level. PMID:23740992

CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.

PubMed

Soh, Chew-Li; Huangfu, Danwei

2017-01-01

The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

PubMed Central

Aebersold, Mathias J.; Thompson-Steckel, Greta; Joutang, Adriane; Schneider, Moritz; Burchert, Conrad; Forró, Csaba; Weydert, Serge; Han, Hana; Vörös, János

2018-01-01

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development. PMID:29535595

Enrichment of spinal cord cell cultures with motoneurons

PubMed Central

1978-01-01

Spinal cord cell cultures contain several types of neurons. Two methods are described for enriching such cultures with motoneurons (defined here simply as cholinergic cells that are capable of innervating muscle). In the first method, 7-day embryonic chick spinal cord neurons were separated according to size by 1 g velocity sedimentation. It is assumed that cholinergic motoneurons are among the largest cells present at this stage. The spinal cords were dissociated vigorously so that 95-98% of the cells in the initial suspension were isolated from one another. Cells in leading fractions (large cell fractions: LCFs) contain about seven times as much choline acetyltransferase (CAT) activity per unit cytoplasm as do cells in trailing fractions (small cell fractions: SCFs). Muscle cultures seeded with LCFs develop 10-70 times as much CAT as cultures seeded with SCFs and six times as much CAT as cultures seeded with control (unfractionated) spinal cord cells. More than 20% of the large neurons in LCF-muscle cultures innervate nearby myotubes. In the second method, neurons were gently dissociated from 4-day embryonic spinal cords and maintained in vitro. This approach is based on earlier observations that cholinergic neurons are among the first cells to withdraw form the mitotic cycle in the developing chick embryo (Hamburger, V. 1948. J. Comp. Neurol. 88:221- 283; and Levi-Montalcini, R. 1950. J. Morphol. 86:253-283). 4-Day spinal cord-muscle cultures develop three times as much CAT as do 7-day spinal cord-muscle plates, prepared in the same (gentle) manner. More than 50% of the relatively large 4-day neurons innervate nearby myotubes. Thus, both methods are useful first steps toward the complete isolation of motoneurons. Both methods should facilitate study of the development of cholinergic neurons and of nerve-muscle synapse formation. PMID:566275

A mathematical model and computational framework for three-dimensional chondrocyte cell growth in a porous tissue scaffold placed inside a bi-directional flow perfusion bioreactor.

PubMed

Shakhawath Hossain, Md; Bergstrom, D J; Chen, X B

2015-12-01

The in vitro chondrocyte cell culture for cartilage tissue regeneration in a perfusion bioreactor is a complex process. Mathematical modeling and computational simulation can provide important insights into the culture process, which would be helpful for selecting culture conditions to improve the quality of the developed tissue constructs. However, simulation of the cell culture process is a challenging task due to the complicated interaction between the cells and local fluid flow and nutrient transport inside the complex porous scaffolds. In this study, a mathematical model and computational framework has been developed to simulate the three-dimensional (3D) cell growth in a porous scaffold placed inside a bi-directional flow perfusion bioreactor. The model was developed by taking into account the two-way coupling between the cell growth and local flow field and associated glucose concentration, and then used to perform a resolved-scale simulation based on the lattice Boltzmann method (LBM). The simulation predicts the local shear stress, glucose concentration, and 3D cell growth inside the porous scaffold for a period of 30 days of cell culture. The predicted cell growth rate was in good overall agreement with the experimental results available in the literature. This study demonstrates that the bi-directional flow perfusion culture system can enhance the homogeneity of the cell growth inside the scaffold. The model and computational framework developed is capable of providing significant insight into the culture process, thus providing a powerful tool for the design and optimization of the cell culture process. © 2015 Wiley Periodicals, Inc.

In-depth analysis of switchable glycerol based polymeric coatings for cell sheet engineering.

PubMed

Becherer, Tobias; Heinen, Silke; Wei, Qiang; Haag, Rainer; Weinhart, Marie

2015-10-01

Scaffold-free cell sheet engineering using thermoresponsive substrates provides a promising alternative to conventional tissue engineering which in general employs biodegradable scaffold materials. We have previously developed a thermoresponsive coating with glycerol based linear copolymers that enables gentle harvesting of entire cell sheets. In this article we present an in-depth analysis of these thermoresponsive linear polyglycidyl ethers and their performance as coating for substrates in cell culture in comparison with commercially available poly(N-isopropylacrylamide) (PNIPAM) coated culture dishes. A series of copolymers of glycidyl methyl ether (GME) and glycidyl ethyl ether (EGE) was prepared in order to study their thermoresponsive properties in solution and on the surface with respect to the comonomer ratio. In both cases, when grafted to planar surfaces or spherical nanoparticles, the applied thermoresponsive polyglycerol coatings render the respective surfaces switchable. Protein adsorption experiments on copolymer coated planar surfaces with surface plasmon resonance (SPR) spectroscopy reveal the ability of the tested thermoresponsive coatings to be switched between highly protein resistant and adsorptive states. Cell culture experiments demonstrate that these thermoresponsive coatings allow for adhesion and proliferation of NIH 3T3 fibroblasts comparable to TCPS and faster than on PNIPAM substrates. Temperature triggered detachment of complete cell sheets from copolymer coated substrates was accomplished within minutes while maintaining high viability of the harvested cells. Thus such glycerol based copolymers present a promising alternative to PNIPAM as a thermoresponsive coating of cell culture substrates. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Monitoring of live cell cultures during apoptosis by phase imaging and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Sharikova, Anna; Saide, George; Sfakis, Lauren; Park, Jun Yong; Desta, Habben; Maloney, Maxwell C.; Castracane, James; Mahajan, Supriya D.; Khmaladze, Alexander

2017-02-01

Non-invasive live cell measurements are an important tool in biomedical research. We present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy records an interference pattern between object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information about the sample. When the phase is mapped across the sample and converted into height information for each pixel, a three dimensional image is obtained. The measurement of live cell cultures by digital holographic microscopy yields information about cell shape and volume, changes to which are reflective of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy, on the other hand, is sensitive to rotational and vibrational molecular transitions, as well as intermolecular vibrations. Therefore, Raman spectroscopy provides complementary information about cells, such as protein, lipid and nucleic acid content, and, particularly, the spectral signatures associated with structural changes in molecules. The cell cultures are kept in the temperature-controlled environmental chamber during the experiment, which allows monitoring over multiple cell cycles. The DHM system combines a visible (red) laser source with conventional microscope base, and LabVIEW-run data processing. We analyzed and compared cell culture information obtained by these two methods.

The costs of using unauthenticated, over-passaged cell lines: how much more data do we need?

PubMed

Hughes, Peyton; Marshall, Damian; Reid, Yvonne; Parkes, Helen; Gelber, Cohava

2007-11-01

Increasing data demonstrate that cellular cross-contamination, misidentified cell lines, and the use of cultures at high-passage levels contribute to the generation of erroneous and misleading results as well as wasted research funds. Contamination of cell lines by other lines has been recognized and documented back to the 1950s. Based on submissions to major cell repositories in the last decade, it is estimated that between 18% and 36% of cell lines may be contaminated or misidentified. More recently, problems surrounding practices of over-subculturing cells are being identified. As a result of selective pressures and genetic drift, cell lines, when kept in culture too long, exhibit reduced or altered key functions and often no longer represent reliable models of their original source material. A review of papers showing significant experimental variances between low- and high-passage cell culture numbers, as well as contaminated lines, makes a strong case for using verified, tested cell lines at low- or defined passage numbers. In the absence of cell culture guidelines, mandates from the National Institutes of Health (NIH) and other funding agencies or journal requirements, it becomes the responsibility of the scientific community to perform due diligence to ensure the integrity of cell cultures used in research.

Thermo-responsive cell culture carrier: Effects on macrophage functionality and detachment efficiency.

PubMed

Rennert, Knut; Nitschke, Mirko; Wallert, Maria; Keune, Natalie; Raasch, Martin; Lorkowski, Stefan; Mosig, Alexander S

2017-01-01

Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte-derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte-derived macrophages. In summary, we observed similar functionality and viability of primary monocyte-derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications.

Development of Fundamental Technologies for Micro Bioreactors

NASA Astrophysics Data System (ADS)

Sato, Kiichi; Kitamori, Takehiko

This chapter reviews the development of fundamental technologies required for microchip-based bioreactors utilizing living mammalian cells and pressure driven flow. The most important factor in the bioreactor is the cell culture. For proper cell culturing, continuous medium supply from a microfluidic channel and appropriate modification of the channel surface to accommodate cell attachment is required. Moreover, the medium flow rate should be chosen carefully, because shear stress affects cell activity. The techniques presented here could be applied to the development of micro bioreactors such as microlivers, pigment production by plant cells, and artificial insemination.

Crosstalk between immune cells and mesenchymal stromal cells in a 3D bioreactor system.

PubMed

Seifert, Martina; Lubitz, Annika; Trommer, Jeanne; Könnig, Darja; Korus, Gabriela; Marx, Uwe; Volk, Hans-Dieter; Duda, Georg; Kasper, Grit; Lehmann, Kerstin; Stolk, Meaghan; Giese, Christoph

2012-11-01

Mesenchymal stromal cells (MSC), known for their high immune modulatory capacity are promising tools for several cell-based therapies. To better mimic the in vivo situation of MSC interactions with immune cells, we applied an artificial lymph node (ALN)-bioreactor culture system combining a miniaturized perfusion bioreactor with a 3D matrix-based cell culture of immune competent cells forming micro-organoids. Rat lymph node cells and allogeneic bone marrow-derived MSCs were seeded in a 20:1 ratio within the agarose matrix of the ALN-reactor. Lymphocytes were pre-incubated with Concanavalin A (ConA) and then co-cultured with MSC in the matrix with additional ConA in the perfusing medium. Live/dead staining showed survival of the co-cultures during the 8-day ALN-reactor run. Paraffin sections of bioreactor matrices were analyzed by proliferating cell nuclear antigen (PCNA)-specific stai-ning to determine MSC proliferation. Immune modulatory capacity was defined by daily analysis of cytokine secretion profiles (TNFa, IFNy, IL-1a, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12p40/p70, GM-CSF). Cytokine peak secretion at day 2 was significantly inhibited by MSCs for TNFa (96.8 ± 4.8%) and IFNy (88.7 ± 12.0%) in 3D co-cultures. In contrast, other cytokines (IL-1, IL-6, IL-12) were induced. Furthermore, we detected a significantly higher (58.8%) fraction of proliferating MSCs in the presence of immune cells compared to control bioreactors loaded with MSCs only. In the future, this system might be an excellent tool to investigate the mechanisms of MSC-mediated immune modulation during simulated in vivo conditions.

Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

PubMed

Terrasso, Ana Paula; Silva, Ana Carina; Filipe, Augusto; Pedroso, Pedro; Ferreira, Ana Lúcia; Alves, Paula Marques; Brito, Catarina

Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates. Copyright © 2016 Elsevier Inc. All rights reserved.

Digital hydraulic drive for microfluidics and miniaturized cell culture devices based on shape memory alloy actuators

NASA Astrophysics Data System (ADS)

Tsai, Cheng-Han; Wu, Xuanye; Kuan, Da-Han; Zimmermann, Stefan; Zengerle, Roland; Koltay, Peter

2018-08-01

In order to culture and analyze individual living cells, microfluidic cultivation and manipulation of cells become an increasingly important topic. Such microfluidic systems allow for exploring the phenotypic differences between thousands of genetically identical cells or pharmacological tests in parallel, which is impossible to achieve by traditional macroscopic cell culture methods. Therefore, plenty of microfluidic systems and devices have been developed for cell biological studies like cell culture, cell sorting, and cell lysis in the past. However, these microfluidic systems are still limited by the external pressure sources which most of the time are large in size and have to be connected by fluidic tubing leading to complex and delicate systems. In order to provide a miniaturized, more robust actuation system a novel, compact and low power consumption digital hydraulic drive (DHD) has been developed that is intended for use in portable and automated microfluidic systems for various applications. The DHD considered in this work consists of a shape memory alloy (SMA) actuator and a pneumatic cylinder. The switching time of the digital modes (pressure ON versus OFF) can be adjusted from 1 s to min. Thus, the DHDs might have many applications for driving microfluidic devices. In this work, different implementations of DHDs are presented and their performance is characterized by experiments. In particular, it will be shown that DHDs can be used for microfluidic large-scale integration (mLSI) valve control (256 valves in parallel) as well as potentially for droplet-based microfluidic systems. As further application example, high-throughput mixing of cell cultures (96 wells in parallel) is demonstrated employing the DHD to drive a so-called ‘functional lid’ (FL), to enable a miniaturized micro bioreactor in a regular 96-well micro well plate.

Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

PubMed Central

Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

2016-01-01

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816

Identification of Regulatory Factors for Mesenchymal Stem Cell-Derived Salivary Epithelial Cells in a Co-Culture System

PubMed Central

Park, Yun-Jong; Koh, Jin; Gauna, Adrienne E.; Chen, Sixue; Cha, Seunghee

2014-01-01

Patients with Sjögren’s syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia. PMID:25402494

Derivation of therapeutic lung spheroid cells from minimally invasive transbronchial pulmonary biopsies.

PubMed

Dinh, Phuong-Uyen C; Cores, Jhon; Hensley, M Taylor; Vandergriff, Adam C; Tang, Junnan; Allen, Tyler A; Caranasos, Thomas G; Adler, Kenneth B; Lobo, Leonard J; Cheng, Ke

2017-06-30

Resident stem and progenitor cells have been identified in the lung over the last decade, but isolation and culture of these cells remains a challenge. Thus, although these lung stem and progenitor cells provide an ideal source for stem-cell based therapy, mesenchymal stem cells (MSCs) remain the most popular cell therapy product for the treatment of lung diseases. Surgical lung biopsies can be the tissue source but such procedures carry a high risk of mortality. In this study we demonstrate that therapeutic lung cells, termed "lung spheroid cells" (LSCs) can be generated from minimally invasive transbronchial lung biopsies using a three-dimensional culture technique. The cells were then characterized by flow cytometry and immunohistochemistry. Angiogenic potential was tested by in-vitro HUVEC tube formation assay. In-vivo bio- distribution of LSCs was examined in athymic nude mice after intravenous delivery. From one lung biopsy, we are able to derive >50 million LSC cells at Passage 2. These cells were characterized by flow cytometry and immunohistochemistry and were shown to represent a mixture of lung stem cells and supporting cells. When introduced systemically into nude mice, LSCs were retained primarily in the lungs for up to 21 days. Here, for the first time, we demonstrated that direct culture and expansion of human lung progenitor cells from pulmonary tissues, acquired through a minimally invasive biopsy, is possible and straightforward with a three-dimensional culture technique. These cells could be utilized in long-term expansion of lung progenitor cells and as part of the development of cell-based therapies for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

Suspension cell culture in microgravity and development of a space bioreactor

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1987-01-01

NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first space bioreactor has been designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small (500 ml) bioreactor is being constructed for flight experiments in the Shuttle middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption, and control of low shear stress on cells.

Integrated strain array for cellular mechanobiology studies

NASA Astrophysics Data System (ADS)

Simmons, C. S.; Sim, J. Y.; Baechtold, P.; Gonzalez, A.; Chung, C.; Borghi, N.; Pruitt, B. L.

2011-05-01

We have developed an integrated strain array for cell culture enabling high-throughput mechano-transduction studies. Biocompatible cell culture chambers were integrated with an acrylic pneumatic compartment and microprocessor-based control system. Each element of the array consists of a deformable membrane supported by a cylindrical pillar within a well. For user-prescribed waveforms, the annular region of the deformable membrane is pulled into the well around the pillar under vacuum, causing the pillar-supported region with cultured cells to be stretched biaxially. The optically clear device and pillar-based mechanism of operation enables imaging on standard laboratory microscopes. Straightforward fabrication utilizes off-the-shelf components, soft lithography techniques in polydimethylsiloxane and laser ablation of acrylic sheets. Proof of compatibility with basic biological assays and standard imaging equipment were accomplished by straining C2C12 skeletal myoblasts on the device for 6 h. At higher strains, cells and actin stress fibers realign with a circumferential preference.

Near-IR laser-triggered target cell collection using a carbon nanotube-based cell-cultured substrate.

PubMed

Sada, Takao; Fujigaya, Tsuyohiko; Niidome, Yasuro; Nakazawa, Kohji; Nakashima, Naotoshi

2011-06-28

Unique near-IR optical properties of single-walled carbon nanotube (SWNTs) are of interest in many biological applications. Here we describe the selective cell detachment and collection from an SWNT-coated cell-culture dish triggered by near-IR pulse laser irradiation. First, HeLa cells were cultured on an SWNT-coated dish prepared by a spraying of an aqueous SWNT dispersion on a glass dish. The SWNT-coated dish was found to show a good cell adhesion behavior as well as a cellular proliferation rate similar to a conventional glass dish. We discovered, by near-IR pulse laser irradiation (at the laser power over 25 mW) to the cell under optical microscopic observation, a quick single-cell detachment from the SWNT-coated surface. Shockwave generation from the irradiated SWNTs is expected to play an important role for the cell detachment. Moreover, we have succeeded in catapulting the target single cell from the cultured medium when the depth of the medium was below 150 μm and the laser power was stronger than 40 mW. The captured cell maintained its original shape. The retention of the genetic information of the cell was confirmed by the polymerase chain reaction (PCR) technique. A target single-cell collection from a culture medium under optical microscopic observation is significant in wide fields of single-cell studies in biological areas.

Animal-cell culture media: History, characteristics, and current issues.

PubMed

Yao, Tatsuma; Asayama, Yuta

2017-04-01

Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.

Nanomagnetic Levitation 3-D Cultures of Breast and Colorectal Cancers

PubMed Central

Bumpers, Harvey L.; Janagama, Dasharatham G.; Manne, Upender; Basson, Marc D.; Katkoori, Venkat

2014-01-01

Background Innovative technologies for drug discovery and development, cancer models, stem cell research, tissue engineering, and drug testing in various cell-based platforms require an application similar to the in vivo system. Materials and Methods We developed for the first time nanomagnetically levitated three dimensional (3-D) cultures of breast cancer (BC) and colorectal cancer (CRC) cells using carbon encapsulated cobalt magnetic nanoparticles. BC and CRC xenografts grown in severe combined immunodeficient (SCID) mice were evaluated for N-cadherin and Epidermal growth factor receptor (EGFR) expressions. These phenotypes were compared with 2-D cultures and 3-D cultures grown in a gel matrix. Results The BC and CRC cells grown by magnetic levitation formed microtissues. The levitated cultures had high viability and were maintained in culture for long periods of time. It has been observed that N-cadherin and EGFR activities were highly expressed in the levitated 3-D tumor spheres and xenografts of CRC and BC cells. Conclusions Nanomagnetically levitated 3-D cultures tend to form stable microtissues of BC and CRC and may be more feasible for a range of applications in drug discovery or regenerative medicine. PMID:25617973

Co-culture systems-based strategies for articular cartilage tissue engineering.

PubMed

Zhang, Yu; Guo, Weimin; Wang, Mingjie; Hao, Chunxiang; Lu, Liang; Gao, Shuang; Zhang, Xueliang; Li, Xu; Chen, Mingxue; Li, Penghao; Jiang, Peng; Lu, Shibi; Liu, Shuyun; Guo, Quanyi

2018-03-01

Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical, and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering. © 2017 Wiley Periodicals, Inc.

Thermoresponsive, in situ crosslinkable hydrogels based on N-isopropylacrylamide: Fabrication, characterization and mesenchymal stem cell encapsulation

PubMed Central

Klouda, Leda; Perkins, Kevin R.; Watson, Brendan M.; Hacker, Michael C.; Bryant, Stephanie J.; Raphael, Robert M.; Kasper, F. Kurtis; Mikos, Antonios G.

2011-01-01

Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently crosslinkable moieties in the macromers. The effects of the macromer end group, namely acrylate or methacrylate, and the fabrication conditions were investigated on the degradative and swelling properties of the hydrogels. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture media at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over eight weeks, with methacrylated hydrogels having higher swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture media under the same conditions showed lower swelling as compared to phosphate buffered saline. The interplay between chemical crosslinking and thermally induced phase separation affected the swelling characteristics of hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over three weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and β-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering. PMID:21187170

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

PubMed

Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

2015-03-01

This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

Detection of cancerous cervical cells using physical adhesion of fluorescent silica particles and centripetal force.

PubMed

Gaikwad, Ravi M; Dokukin, Maxim E; Iyer, K Swaminathan; Woodworth, Craig D; Volkov, Dmytro O; Sokolov, Igor

2011-04-07

Here we describe a non-traditional method to identify cancerous human cervical epithelial cells in a culture dish based on physical adhesion between silica beads and cells. It is a simple optical fluorescence-based technique which detects the relative difference in the amount of fluorescent silica beads physically adherent to surfaces of cancerous and normal cervical cells. The method utilizes the centripetal force gradient that occurs in a rotating culture dish. Due to the variation in the balance between adhesion and centripetal forces, cancerous and normal cells demonstrate clearly distinctive distributions of the fluorescent particles adherent to the cell surface over the culture dish. The method demonstrates higher adhesion of silica particles to normal cells compared to cancerous cells. The difference in adhesion was initially observed by atomic force microscopy (AFM). The AFM data were used to design the parameters of the rotational dish experiment. The optical method that we describe is much faster and technically simpler than AFM. This work provides proof of the concept that physical interactions can be used to accurately discriminate normal and cancer cells. © The Royal Society of Chemistry 2011

Quantification of substrate and cellular strains in stretchable 3D cell cultures: an experimental and computational framework.

PubMed

González-Avalos, P; Mürnseer, M; Deeg, J; Bachmann, A; Spatz, J; Dooley, S; Eils, R; Gladilin, E

2017-05-01

The mechanical cell environment is a key regulator of biological processes . In living tissues, cells are embedded into the 3D extracellular matrix and permanently exposed to mechanical forces. Quantification of the cellular strain state in a 3D matrix is therefore the first step towards understanding how physical cues determine single cell and multicellular behaviour. The majority of cell assays are, however, based on 2D cell cultures that lack many essential features of the in vivo cellular environment. Furthermore, nondestructive measurement of substrate and cellular mechanics requires appropriate computational tools for microscopic image analysis and interpretation. Here, we present an experimental and computational framework for generation and quantification of the cellular strain state in 3D cell cultures using a combination of 3D substrate stretcher, multichannel microscopic imaging and computational image analysis. The 3D substrate stretcher enables deformation of living cells embedded in bead-labelled 3D collagen hydrogels. Local substrate and cell deformations are determined by tracking displacement of fluorescent beads with subsequent finite element interpolation of cell strains over a tetrahedral tessellation. In this feasibility study, we debate diverse aspects of deformable 3D culture construction, quantification and evaluation, and present an example of its application for quantitative analysis of a cellular model system based on primary mouse hepatocytes undergoing transforming growth factor (TGF-β) induced epithelial-to-mesenchymal transition. © 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Hand-made cloned goat (Capra hircus) embryos—a comparison of different donor cells and culture systems.

PubMed

Akshey, Yogesh S; Malakar, Dhruba; De, Arun K; Jena, Manoj K; Garg, Shweta; Dutta, Rahul; Pawar, Sachin Kumar; Mukesh, Manisha

2010-10-01

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells

PubMed Central

Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin; Brogaard, Louise; Flagstad, Frederikke Bjergvang; Emnéus, Jenny; Wolff, Anders; Summerfield, Artur; Jungersen, Gregers

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today. PMID:27362493

Investigating the Role of Surface Materials and Three Dimensional Architecture on In Vitro Differentiation of Porcine Monocyte-Derived Dendritic Cells.

PubMed

Hartmann, Sofie Bruun; Mohanty, Soumyaranjan; Skovgaard, Kerstin; Brogaard, Louise; Flagstad, Frederikke Bjergvang; Emnéus, Jenny; Wolff, Anders; Summerfield, Artur; Jungersen, Gregers

2016-01-01

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

PubMed

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-04-03

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants.

PubMed

Paul, Albert Jesuran; Schwab, Karen; Prokoph, Nina; Haas, Elena; Handrick, René; Hesse, Friedemann

2015-06-01

Product yields, efficacy, and safety of monoclonal antibodies (mAbs) are reduced by the formation of higher molecular weight aggregates during upstream processing. In-process characterization of mAb aggregate formation is a challenge since there is a lack of a fast detection method to identify mAb aggregates in cell culture. In this work, we present a rapid method to characterize mAb aggregate-containing Chinese hamster ovary (CHO) cell culture supernatants. The fluorescence dyes thioflavin T (ThT) and 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS) enabled the detection of soluble as well as large mAb aggregates. Partial least square (PLS) regression models were used to evaluate the linearity of the dye-based mAb aggregate detection in buffer down to a mAb aggregate concentration of 2.4 μg mL(-1). Furthermore, mAb aggregates were detected in bioprocess medium using Bis-ANS and ThT. Dye binding to aggregates was stable for 60 min, making the method robust and reliable. Finally, the developed method using 10 μmol L(-1) Bis-ANS enabled discrimination between CHO cell culture supernatants containing different levels of mAb aggregates. The method can be adapted for high-throughput screening, e.g., to screen for cell culture conditions influencing mAb product quality, and hence can contribute to the improvement of production processes of biopharmaceuticals in mammalian cell culture.

A Contact-Based Method for Differentiation of Human Mesenchymal Stem Cells into an Endothelial Cell-Phenotype.

PubMed

Joddar, Binata; Kumar, Shweta Anil; Kumar, Alok

2018-06-01

Adult stem cells such as mesenchymal stem cells (MSC) are known to possess the ability to augment neovascularization processes and are thus widely popular as an autologous source of progenitor cells. However there is a huge gap in our current knowledge of mechanisms involved in differentiating MSC into endothelial cells (EC), essential for lining engineered blood vessels. To fill up this gap, we attempted to differentiate human MSC into EC, by culturing the former onto chemically fixed layers of EC or its ECM, respectively. We expected direct contact of MSC when cultured atop fixed EC or its ECM, would coax the former to differentiate into EC. Results showed that human MSC cultured atop chemically fixed EC or its ECM using EC-medium showed enhanced expression of CD31, a marker for EC, compared to other cases. Further in all human MSC cultured using EC-medium, typically characteristic cobble stone shaped morphologies were noted in comparison to cells cultured using MSC medium, implying that the differentiated cells were sensitive to soluble VEGF supplementation present in the EC-medium. Results will enhance and affect therapies utilizing autologous MSC as a cell source for generating vascular cells to be used in a variety of tissue engineering applications.

Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

PubMed

Liew, Lawrence J; Day, Richard M; Dilley, Rodney J

2017-03-01

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PubMed

Ida, Chieri; Yamashita, Sachiko; Tsukada, Masaki; Sato, Teruaki; Eguchi, Takayuki; Tanaka, Masakazu; Ogata, Shin; Fujii, Takahiro; Nishi, Yoshisuke; Ikegami, Susumu; Moss, Joel; Miwa, Masanao

2016-02-01

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

PubMed

Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

2015-06-01

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

PubMed Central

Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P.

2015-01-01

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue™, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required. PMID:25336207

Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids

PubMed Central

Weegman, Bradley P.; Nash, Peter; Carlson, Alexandra L.; Voltzke, Kristin J.; Geng, Zhaohui; Jahani, Marjan; Becker, Benjamin B.; Papas, Klearchos K.; Firpo, Meri T.

2013-01-01

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications. PMID:24204645

Optimization of insect cell based protein production processes - online monitoring, expression systems, scale up.

PubMed

Druzinec, Damir; Salzig, Denise; Brix, Alexander; Kraume, Matthias; Vilcinskas, Andreas; Kollewe, Christian; Czermak, Peter

2013-01-01

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes.

Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

PubMed

Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

2014-01-01

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

Image analysis supported moss cell disruption in photo-bioreactors.

PubMed

Lucumi, A; Posten, C; Pons, M-N

2005-05-01

Diverse methods for the disruption of cell entanglements and pellets of the moss Physcomitrella patens were tested in order to improve the homogeneity of suspension cultures. The morphological characterization of the moss was carried out by means of image analysis. Selected morphological parameters were defined and compared to the reduction of the carbon dioxide fixation, and the released pigments after cell disruption. The size control of the moss entanglements based on the rotor stator principle allowed a focused shear stress, avoiding a severe reduction in the photosynthesis. Batch cultures of P. patens in a 30.0-l pilot tubular photo-bioreactor with cell disruption showed no significant variation in growth rate and a delayed cell differentiation, when compared to undisrupted cultures. A highly controlled photoautotrophic culture of P. patens in a scalable photo-bioreactor was established, contributing to the development required for the future use of mosses as producers of relevant heterologous proteins.

Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

PubMed

Huang, Guo-Shiang; Tseng, Ting-Chen; Dai, Niann-Tzyy; Fu, Keng-Yen; Dai, Lien-Guo; Hsu, Shan-Hui

2015-10-01

Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures

PubMed Central

Nakano, Tomoyuki; Kanai, Yoshihiko; Amano, Yusuke; Yoshimoto, Taichiro; Matsubara, Daisuke; Shibano, Tomoki; Tamura, Tomoko; Oguni, Sachiko; Katashiba, Shizuka; Ito, Takeshi; Murakami, Yoshinori; Fukayama, Masashi; Murakami, Takashi; Endo, Shunsuke; Niki, Toshiro

2017-01-01

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3–4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion. PMID:28786996

Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers.

PubMed Central

Sutherland, H J; Lansdorp, P M; Henkelman, D H; Eaves, A C; Eaves, C J

1990-01-01

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report, we show that such cultures can be used to provide a quantitative assay for human "LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met, the clonogenic cell output (determined by assessment of 5-week-old cultures) is linearly related to the input cell number over a wide range of cell concentrations. Using limiting dilution analysis techniques, we have established the frequency of LTC-initiating cells in normal human marrow to be approximately 1 per 2 X 10(4) cells and in a highly purified CD34-positive subpopulation to be approximately 1 per 50-100 cells. The proliferative capacity exhibited by individual LTC-initiating cells cultured under apparently identical culture conditions was found to be highly variable. Values for the number of clonogenic cells per LTC-initiating cell in 5-week-old cultures ranged from 1 to 30 (the average being 4) with similar levels being detected in positive 8-week-old cultures. Some LTC-initiating cells are multipotent as evidenced by their generation of erythroid as well as granulopoietic progeny. The availability of a system for quantitative analysis of the proliferative and differentiative behavior of this newly defined compartment of primitive human hematopoietic cells should facilitate future studies of specific genetic or microenvironmental parameters involved in the regulation of these cells. Images PMID:2333304

Methods for Incorporating Oxygen-Generating Biomaterials into Cell Culture and Microcapsule Systems.

PubMed

McQuilling, John Patrick; Opara, Emmanuel C

2017-01-01

A major obstacle to long-term performance of tissue construct implants in regenerative medicine is the inherent hypoxia to which cells in the engineered construct are exposed prior to vascularization of the implant. Various approaches are currently being designed to address this problem. An emerging area of interest on this issue is the use of peroxide-based materials to generate oxygen during the critical period of extended hypoxia that occurs from the time cells are in culture waiting to be used in tissue engineering devices through the immediate post-implant period. In this chapter we provide protocols that we have developed for using these chemical oxygen generators in cell culture and tissue constructs as illustrated by pancreatic islet cell microencapsulation.

Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

PubMed

Nampe, Daniel; Joshi, Ronak; Keller, Kevin; Zur Nieden, Nicole I; Tsutsui, Hideaki

2017-09-01

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics.

PubMed

Fitzgerald, Kathleen A; Guo, Jianfeng; Tierney, Erica G; Curtin, Caroline M; Malhotra, Meenakshi; Darcy, Raphael; O'Brien, Fergal J; O'Driscoll, Caitriona M

2015-10-01

Prostate cancer bone metastases are a leading cause of cancer-related death in men with current treatments offering only marginally improved rates of survival. Advances in the understanding of the genetic basis of prostate cancer provide the opportunity to develop gene-based medicines capable of treating metastatic disease. The aim of this work was to establish a 3D cell culture model of prostate cancer bone metastasis using collagen-based scaffolds, to characterise this model, and to assess the potential of the model to evaluate delivery of gene therapeutics designed to target bone metastases. Two prostate cancer cell lines (PC3 and LNCaP) were cultured in 2D standard culture and compared to 3D cell growth on three different collagen-based scaffolds (collagen and composites of collagen containing either glycosaminoglycan or nanohydroxyapatite). The 3D model was characterised for cell proliferation, viability and for matrix metalloproteinase (MMP) enzyme and Prostate Specific Antigen (PSA) secretion. Chemosensitivity to docetaxel treatment was assessed in 2D in comparison to 3D. Nanoparticles (NPs) containing siRNA formulated using a modified cyclodextrin were delivered to the cells on the scaffolds and gene silencing was quantified. Both prostate cancer cell lines actively infiltrated and proliferated on the scaffolds. Cell culture in 3D resulted in reduced levels of MMP1 and MMP9 secretion in PC3 cells. In contrast, LNCaP cells grown in 3D secreted elevated levels of PSA, particularly on the scaffold composed of collagen and glycosaminoglycans. Both cell lines grown in 3D displayed increased resistance to docetaxel treatment. The cyclodextrin.siRNA nanoparticles achieved cellular uptake and knocked down the endogenous GAPDH gene in the 3D model. In conclusion, development of a novel 3D cell culture model of prostate cancer bone metastasis has been initiated resulting, for the first time, in the successful delivery of gene therapeutics in a 3D in vitro model. Further enhancement of this model will help elucidate the pathogenesis of prostate cancer and also accelerate the design of effective therapies which can penetrate into the bone microenvironment for prostate cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

A unique noninvasive approach to monitoring dissolved O2 and CO2 in cell culture.

PubMed

Chatterjee, Madhubanti; Ge, Xudong; Uplekar, Shaunak; Kostov, Yordan; Croucher, Leah; Pilli, Manohar; Rao, Govind

2015-01-01

Although online monitoring of dissolved oxygen (DO) and carbon dioxide (DCO2 ) is highly desirable in bioprocesses, small-scale bioreactors are usually not monitored due to the lack of suitable sensors. Traditional electrochemical sensors are usually not used because they are bulky and invasive. Disposable optical sensors are small and only partially invasive, but there are concerns regarding the toxicity of the patch and the phototoxicity of the illuminating light. Here we present a novel, noninvasive, rate-based technique for monitoring DO and DCO2 in cell cultures. A silicone sampling loop which allowed the diffusion of O2 and CO2 through its wall was inserted inside a bioreactor, and then flushed with N2 until the CO2 and O2 inside the loop were completely removed. The gas inside the loop was then allowed to recirculate through gas impermeable tubing to the O2 and CO2 sensors. We have shown that by measuring the initial diffusion rate we were able to determine the partial pressures of the two gases in the culture. The technique could be readily automated and measurements could be made in minutes. It was tested in demonstration experiments by growing murine hybridoma cells in a T-flask and a spinner-flask at 37°C. The results were comparable to those measured with commercially available fluorescence-based patch sensors. These results show that the rate-based method is an effective way to monitor small-scale cell cultures. This measurement mechanism can be easily built into disposable cell culture vessels for facile use. © 2014 Wiley Periodicals, Inc.

Mathematical modeling of the flow field and particle motion in a rotating bioreactor at unit gravity and microgravity

NASA Technical Reports Server (NTRS)

Boyd, Ernest J.

1990-01-01

The biotechnology group at NASA Johnson Space Center is developing systems for culturing mammalian cells that stimulate some aspect of microgravity and provide a low shear environment for microgravity-based studies on suspension and anchorage dependent cells. The design of these vessels for culturing cells is based on the need to suspend cells and aggregates of cells and microcarrier beads continually in the culturing medium. The design must also provide sufficient circulation for adequate mass transfer of nutrients to the cells and minimize the total force on the cells. Forces, resulting from sources such as hydrodynamic fluid shear and collisions of cells and walls of the vessels, may damage delicate cells and degrade the formation of three dimensional structures. This study examines one particular design in both unit gravity and microgravity based on two concentric cylinders rotating in the same direction at different speeds to create a Couette flow between them. A numerical simulation for the flow field and the trajectories of particles in the vessel. The flow field for the circulation of the culturing medium is modeled by the Navier-Stokes equations. The forces on a particle are assumed to be drag from the fluid's circulation, buoyancy from the gravitational force and centrifugal force from the rotation of the vessel. The problem requires first solving the system of partial differential equations for the fluid flow by a finite difference method and then solving the system of ordinary differential equations for the trajectories by Gear's stiff method. Results of the study indicate that the trajectories in unit gravity and microgravity are very similar except for small spatial deviations on the fast time scale in unit gravity. The total force per unit cross sectional area on a particle in microgravity, however, is significantly smaller than the corresponding value in unit gravity, which is also smaller than anticipated. Hence, this study indicates that this design for a bioreactor with optimal rates of rotation can provide a good environment for culturing cells in microgravity with adequate circulation and minimal force on the cells.

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine.

PubMed

Murabayashi, Dai; Mochizuki, Mai; Tamaki, Yuichi; Nakahara, Taka

2017-07-01

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.

Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

PubMed Central

Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh

2016-01-01

Abstract Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell−1 s−1, and 5 and 35 amol cell−1 s−1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies. PMID:27214658

Cell culture-based tissue engineering as an alternative to bone grafts in implant dentistry: a literature review.

PubMed

Boeckel, Daniel Gonçalves; Shinkai, Rosemary Sadami Arai; Grossi, Márcio Lima; Teixeira, Eduardo Rolim

2012-09-01

Several biomaterials and techniques for bone grafting have been described in the literature for atresic bone tissue replacement caused by edentulism, surgical resectioning, and traumas. A new technique involves tissue engineering, a promising option to replace bone tissue and solve problems associated with morbidity of autogenous grafting. This literature review aims to describe tissue-engineering techniques using ex vivo cell culture as an alternative to repair bone maxillary atresias and discuss the concepts and potentials of bone regeneration through cell culture techniques as an option for restorative maxillofacial surgery.

Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells.

PubMed

Ohlsson, Lars; Exley, Christopher; Darabi, Anna; Sandén, Emma; Siesjö, Peter; Eriksson, Håkan

2013-11-01

Aluminium oxyhydroxide, Al(OH)3 is one of few compounds approved as an adjuvant in human vaccines. However, the mechanism behind its immune stimulating properties is still poorly understood. In vitro co-culture of an aluminium adjuvant and the human monocytic cell line THP-1 resulted in reduced cell proliferation. Inhibition occurred at concentrations of adjuvant several times lower than would be found at the injection site using a vaccine formulation containing an aluminium adjuvant. Based on evaluation of the mitochondrial membrane potential, THP-1 cells showed no mitochondrial rupture after co-culture with the aluminium adjuvant, instead an increase in mitochondrial activity was seen. The THP-1 cells are phagocytosing cells and after co-culture with the aluminium adjuvant the phagosomal pathway was obstructed. Primary or early phagosomes mature into phagolysosomes with an internal pH of 4.5 - 5 and carry a wide variety of hydrolysing enzymes. Co-culture with the aluminium adjuvant yielded a reduced level of acidic vesicles and cathepsin L activity, a proteolytic enzyme of the phagolysosomes, was almost completely inhibited. THP-1 cells are an appropriate in vitro model in order to investigate the mechanism behind the induction of a phagocytosing antigen presenting cell into an inflammatory cell by aluminium adjuvants. Much information will be gained by investigating the phagosomal pathway and what occurs inside the phagosomes and to elucidate the ultimate fate of phagocytosed aluminium particles. © 2013.

A quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine.

PubMed

Gregersen, Jens-Peter

2008-06-19

A risk-assessment model has demonstrated the ability of a new cell culture-based vaccine manufacturing process to reduce the level of any adventitious agent to a million-fold below infectious levels. The cell culture-derived subunit influenza vaccine (OPTAFLU), Novartis Vaccines and Diagnostics) is produced using Madin-Darby canine kidney (MDCK) cells to propagate seasonal viral strains, as an alternative to embryonated chicken-eggs. As only a limited range of mammalian viruses can grow in MDCK cells, similar to embryonated eggs, MDCK cells can act as an effective filter for a wide range of adventitious agents that might be introduced during vaccine production. However, the introduction of an alternative cell substrate (for example, MDCK cells) into a vaccine manufacturing process requires thorough investigations to assess the potential for adventitious agent risk in the final product, in the unlikely event that contamination should occur. The risk assessment takes into account the entire manufacturing process, from initial influenza virus isolation, through to blending of the trivalent subunit vaccine and worst-case residual titres for the final vaccine formulation have been calculated for >20 viruses or virus families. Maximum residual titres for all viruses tested were in the range of 10(-6) to 10(-16) infectious units per vaccine dose. Thus, the new cell culture-based vaccine manufacturing process can reduce any adventitious agent to a level that is unable to cause infection.

The role of corneal endothelial morphology in graft assessment and prediction of endothelial cell loss during organ culture of human donor corneas.

PubMed

Hermel, Martin; Salla, Sabine; Fuest, Matthias; Walter, Peter

2017-03-01

Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. I-ECD was 2812 ± 360/mm 2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm 2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm 2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve graft quality. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues.

PubMed

Li, Xingnan; Ootani, Akifumi; Kuo, Calvin

2016-01-01

Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.

Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells.

PubMed

Hu, Suwen; Deng, Lei; Wang, Huamao; Zhuang, Yingping; Chu, Ju; Zhang, Siliang; Li, Zhonghai; Guo, Meijin

2011-05-01

The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 10(5) cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fed-batch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.

Analysis of the Growth Process of Neural Cells in Culture Environment Using Image Processing Techniques

NASA Astrophysics Data System (ADS)

Mirsafianf, Atefeh S.; Isfahani, Shirin N.; Kasaei, Shohreh; Mobasheri, Hamid

Here we present an approach for processing neural cells images to analyze their growth process in culture environment. We have applied several image processing techniques for: 1- Environmental noise reduction, 2- Neural cells segmentation, 3- Neural cells classification based on their dendrites' growth conditions, and 4- neurons' features Extraction and measurement (e.g., like cell body area, number of dendrites, axon's length, and so on). Due to the large amount of noise in the images, we have used feed forward artificial neural networks to detect edges more precisely.

Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

NASA Astrophysics Data System (ADS)

Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

Cell culture in autologous fibrin scaffolds for applications in tissue engineering.

PubMed

de la Puente, Pilar; Ludeña, Dolores

2014-03-10

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth. With this review our aim is to follow methods of development, analyze the commercial and autologous fibrins available and assess the possible applications of cell culture in tissue engineering in these three-dimensional structures. Copyright © 2013 Elsevier Inc. All rights reserved.

Comparative analyses of industrial-scale human platelet lysate preparations.

PubMed

Pierce, Jan; Benedetti, Eric; Preslar, Amber; Jacobson, Pam; Jin, Ping; Stroncek, David F; Reems, Jo-Anna

2017-12-01

Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product. Nine industrial-scale, serum-based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet-rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma-based or serum-based PL were compared to MSCs cultured with fetal bovine serum. Electrolyte and protein levels were relatively consistent among all serum-based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum-based PL stored at -80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum-based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement. Using a large-scale, standardized method, lot-to-lot variations were noted for industrial-scale preparations of serum-based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off-the-shelf PL is a feasible substitute for fetal bovine serum in MSC cultures. © 2017 AABB.

Biomimetic Graphene-Based 3D Scaffold for Long-Term Cell Culture and Real-Time Electrochemical Monitoring.

PubMed

Hu, Xue-Bo; Liu, Yan-Ling; Wang, Wen-Jie; Zhang, Hai-Wei; Qin, Yu; Guo, Shan; Zhang, Xin-Wei; Fu, Lei; Huang, Wei-Hua

2018-01-16

Current achievements on electrochemical monitoring of cells are often gained on two-dimensional (2D) substrates, which fail in mimicking the cellular environments and accurately reproducing the cellular functions within a three-dimensional (3D) tissue. In this regard, 3D scaffold concurrently integrated with the function of cell culture and electrochemical sensing is conceivably a promising platform to monitor cells in real time under their in vivo-like 3D microenvironments. However, it is particularly challenging to construct such a multifunctional scaffold platform. Herein, we developed a 3-aminophenylboronic acid (APBA) functionalized graphene foam (GF) network, which combines the biomimetic property of APBA with the mechanical and electrochemical properties of GF. Hence, the GF network can serve as a 3D scaffold to culture cells for a long period with high viability and simultaneously as an electrode for highly sensitive electrochemical sensing. This allows monitoring of gaseous messengers H 2 S released from the cells cultured on the 3D scaffold in real time. This work represents considerable progress in fabricating 3D cell culture scaffold with electrochemical properties, thereby facilitating future studies of physiologically relevant processes.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

PubMed

Appelt-Menzel, Antje; Cubukova, Alevtina; Günther, Katharina; Edenhofer, Frank; Piontek, Jörg; Krause, Gerd; Stüber, Tanja; Walles, Heike; Neuhaus, Winfried; Metzger, Marco

2017-04-11

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Prediction of Microbial Infection of Cultured Cells Using DNA Microarray Gene-Expression Profiles of Host Responses

PubMed Central

Park, Yu Rang; Chung, Tae Su; Lee, Young Joo; Song, Yeong Wook; Lee, Eun Young; Sohn, Yeo Won; Song, Sukgil; Park, Woong Yang

2012-01-01

Infection by microorganisms may cause fatally erroneous interpretations in the biologic researches based on cell culture. The contamination by microorganism in the cell culture is quite frequent (5% to 35%). However, current approaches to identify the presence of contamination have many limitations such as high cost of time and labor, and difficulty in interpreting the result. In this paper, we propose a model to predict cell infection, using a microarray technique which gives an overview of the whole genome profile. By analysis of 62 microarray expression profiles under various experimental conditions altering cell type, source of infection and collection time, we discovered 5 marker genes, NM_005298, NM_016408, NM_014588, S76389, and NM_001853. In addition, we discovered two of these genes, S76389, and NM_001853, are involved in a Mycolplasma-specific infection process. We also suggest models to predict the source of infection, cell type or time after infection. We implemented a web based prediction tool in microarray data, named Prediction of Microbial Infection (http://www.snubi.org/software/PMI). PMID:23091307

Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor.

PubMed

Rafiq, Qasim A; Hanga, Mariana P; Heathman, Thomas R J; Coopman, Karen; Nienow, Alvin W; Williams, David J; Hewitt, Christopher J

2017-10-01

Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, N JS . The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06-0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253-2266. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

PubMed Central

Hanga, Mariana P.; Heathman, Thomas R. J.; Coopman, Karen; Nienow, Alvin W.; Williams, David J.; Hewitt, Christopher J.

2017-01-01

ABSTRACT Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum‐free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253–2266. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28627713

A durable and biocompatible ascorbic acid-based covalent coating method of polydimethylsiloxane for dynamic cell culture.

PubMed

Leivo, Joni; Virjula, Sanni; Vanhatupa, Sari; Kartasalo, Kimmo; Kreutzer, Joose; Miettinen, Susanna; Kallio, Pasi

2017-07-01

Polydimethylsiloxane (PDMS) is widely used in dynamic biological microfluidic applications. As a highly hydrophobic material, native PDMS does not support cell attachment and culture, especially in dynamic conditions. Previous covalent coating methods use glutaraldehyde (GA) which, however, is cytotoxic. This paper introduces a novel and simple method for binding collagen type I covalently on PDMS using ascorbic acid (AA) as a cross-linker instead of GA. We compare the novel method against physisorption and GA cross-linker-based methods. The coatings are characterized by immunostaining, contact angle measurement, atomic force microscopy and infrared spectroscopy, and evaluated in static and stretched human adipose stem cell (hASC) cultures up to 13 days. We found that AA can replace GA as a cross-linker in the covalent coating method and that the coating is durable after sonication and after 6 days of stretching. Furthermore, we show that hASCs attach and proliferate better on AA cross-linked samples compared with physisorbed or GA-based methods. Thus, in this paper, we provide a new PDMS coating method for studying cells, such as hASCs, in static and dynamic conditions. The proposed method is an important step in the development of PDMS-based devices in cell and tissue engineering applications. © 2017 The Author(s).

SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

PubMed

Arhoma, A; Chantry, A D; Haywood-Small, S L; Cross, N A

2017-11-15

Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell populations that had been selected for TRAIL-resistance from initially TRAIL-sensitive populations. SAHA may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Fabrication of a co-culture micro-bioreactor device for efficient hepatic differentiation of human induced pluripotent stem cells (hiPSCs).

PubMed

Kehtari, Mousa; Zeynali, Bahman; Soleimani, Masoud; Kabiri, Mahboubeh; Seyedjafari, Ehsan

2018-04-27

Primary hepatocytes, as the gold standard cell type for in vitro models, lose their characteristic morphology and functions after few days. There is an urgent need to develop physiologically relevant models that recapitulate liver microenvironment to obtain mature hepatocyte from stem cells. We designed and fabricated a micro-bioreactor device mimicking the physiological shear stress and cell-cell interaction in liver sinusoid microenvironment. Induced pluripotent stem cells (iPSCs) were co-cultured with human umbilical vein endothelial cells (HUVECs) in the micro-bioreactor device with continuous perfusion of hepatic differentiation medium (100 μL/h). Simulation results showed that flow field inside our perfusion device was uniform and shear stress was adjusted to physiological condition (<2 dyne/cm 2 ). IPSCs-derived hepatocytes (iPSCs-Heps) that were cultured in micro-bioreactor device showed a higher level of hepatic markers compared to those in static condition. Flow cytometry and immunocytochemistry analysis revealed iPSCs cultured in the device sequentially acquired characteristics of definitive endodermal cells (SOX17 positive), hepatoblasts (AFP positive) and mature hepatocyte (ALB positive). Moreover, the albumin and urea secretion were significantly higher in micro-bioreactor device than those cultured in culture dishes during experiment. Thus, based on our results, we propose our micro-bioreactor as a beneficial device to generate mature hepatocytes for drug screening and basic research.

Nutritional effects of culture media on mycoplasma cell size and removal by filtration.

PubMed

Folmsbee, Martha; Howard, Glenn; McAlister, Morven

2010-03-01

Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 microm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 microm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 microm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 microm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 microm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 microm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum. (c) 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Enhanced production of enveloped viruses in BST-2-deficient cell lines.

PubMed

Yi, Eunbi; Oh, Jinsoo; Giao, Ngoc Q; Oh, Soohwan; Park, Se-Ho

2017-10-01

Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero-specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV-68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST-2 expression in virus-producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289-2297. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Cell Adhesion on Surface-Functionalized Magnesium.

PubMed

Wagener, Victoria; Schilling, Achim; Mainka, Astrid; Hennig, Diana; Gerum, Richard; Kelch, Marie-Luise; Keim, Simon; Fabry, Ben; Virtanen, Sannakaisa

2016-05-18

The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance.

Improvement of biomaterials used in tissue engineering by an ageing treatment.

PubMed

Acevedo, Cristian A; Díaz-Calderón, Paulo; Enrione, Javier; Caneo, María J; Palacios, Camila F; Weinstein-Oppenheimer, Caroline; Brown, Donald I

2015-04-01

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60% (scaffold without ageing treatment) to 80%. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.

Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

NASA Technical Reports Server (NTRS)

Marquette, Michele L.; Sognier, Marguerite A.

2013-01-01

An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

PubMed

Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh; Szita, Nicolas

2016-09-01

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Clinical application of adipose stem cells in plastic surgery.

PubMed

Kim, Yong-Jin; Jeong, Jae-Ho

2014-04-01

Adipose stem cells (ASCs) are a type of adult stem cells that share common characteristics with typical mesenchymal stem cells. In the last decade, ASCs have been shown to be a useful cell resource for tissue regeneration. The major role of regenerative medicine in this century is based on cell therapy in which ASCs hold a key position. Active research on this new type of adult stem cell has been ongoing and these cells now have several clinical applications, including fat grafting, overcoming wound healing difficulties, recovery from local tissue ischemia, and scar remodeling. The application of cultured cells will increase the efficiency of cell therapy. However, the use of cultured stem cells is strictly controlled by government regulation to ensure patient safety. Government regulation is a factor that can limit more versatile clinical application of ASCs. In this review, current clinical applications of ASCs in plastic surgery are introduced. Future stem cell applications in clinical field including culturing and banking of ASCs are also discussed in this review.

Influence of non-adherent yeast cells on electrical characteristics of diamond-based field-effect transistors

NASA Astrophysics Data System (ADS)

Procházka, Václav; Cifra, Michal; Kulha, Pavel; Ižák, Tibor; Rezek, Bohuslav; Kromka, Alexander

2017-02-01

Diamond thin films provide unique features as substrates for cell cultures and as bio-electronic sensors. Here we employ solution-gated field effect transistors (SGFET) based on nanocrystalline diamond thin films with H-terminated surface which exhibits the sub-surface p-type conductive channel. We study an influence of yeast cells (Saccharomyces cerevisiae) on electrical characteristics of the diamond SGFETs. Two different cell culture solutions (sucrose and yeast peptone dextrose-YPD) are used, with and without the cells. We have found that transfer characteristics of the SGFETs exhibit a negative shift of the gate voltage by -26 mV and -42 mV for sucrose and YPD with cells in comparison to blank solutions without the cells. This effect is attributed to a local pH change in close vicinity of the H-terminated diamond surface due to metabolic processes of the yeast cells. The pH sensitivity of the diamond-based SGFETs, the role of cell and protein adhesion on the gate surface and the role of negative surface charge of yeast cells on the SGFETs electrical characteristics are discussed as well.

Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

PubMed

Wang, Juan; Hao, Jie; Bai, Donghui; Gu, Qi; Han, Weifang; Wang, Lei; Tan, Yuanqing; Li, Xia; Xue, Ke; Han, Pencheng; Liu, Zhengxin; Jia, Yundan; Wu, Jun; Liu, Lei; Wang, Liu; Li, Wei; Liu, Zhonghua; Zhou, Qi

2015-11-12

Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents. Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III". We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. The clinical-grade hiPSC lines therefore could be valuable sources for future hiPSC-based clinical trials or therapies and for drug screening.

In vitro cell culture models to study the corneal drug absorption.

PubMed

Reichl, Stephan; Kölln, Christian; Hahne, Matthias; Verstraelen, Jessica

2011-05-01

Many diseases of the anterior eye segment are treated using topically applied ophthalmic drugs. For these drugs, the cornea is the main barrier to reaching the interior of the eye. In vitro studies regarding transcorneal drug absorption are commonly performed using excised corneas from experimental animals. Due to several disadvantages and limitations of these animal experiments, establishing corneal cell culture models has been attempted as an alternative. This review summarizes the development of in vitro models based on corneal cell cultures for permeation studies during the last 20 years, starting with simple epithelial models and moving toward complex organotypical 3D corneal equivalents. Current human 3D corneal cell culture models have the potential to replace excised animal corneas in drug absorption studies. However, for widespread use, the contemporary validation of existent systems is required.

T cell regulation in microgravity - The current knowledge from in vitro experiments conducted in space, parabolic flights and ground-based facilities

NASA Astrophysics Data System (ADS)

Hauschild, Swantje; Tauber, Svantje; Lauber, Beatrice; Thiel, Cora S.; Layer, Liliana E.; Ullrich, Oliver

2014-11-01

Dating back to the Apollo and Skylab missions, it has been reported that astronauts suffered from bacterial and viral infections during space flight or after returning to Earth. Blood analyses revealed strongly reduced capability of human lymphocytes to become active upon mitogenic stimulation. Since then, a large number of in vitro studies on human immune cells have been conducted in space, in parabolic flights, and in ground-based facilities. It became obvious that microgravity affects cell morphology and important cellular functions. Observed changes include cell proliferation, the cytoskeleton, signal transduction and gene expression. This review gives an overview of the current knowledge of T cell regulation under altered gravity conditions obtained by in vitro studies with special emphasis on the cell culture conditions used. We propose that future in vitro experiments should follow rigorous standardized cell culture conditions, which allows better comparison of the results obtained in different flight- and ground-based experiment platforms.

A chemically defined substrate for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

PubMed

Tsai, Yihuan; Cutts, Josh; Kimura, Azuma; Varun, Divya; Brafman, David A

2015-07-01

Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS), could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i) defined, xeno-free conditions for their expansion and neuronal differentiation and (ii) scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP)-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol) (P4VP), that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery. Copyright © 2015. Published by Elsevier B.V.

Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells.

PubMed

Christakou, Athanasia E; Ohlin, Mathias; Önfelt, Björn; Wiklund, Martin

2015-08-07

We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

Establishment of a fully automated microtiter plate-based system for suspension cell culture and its application for enhanced process optimization.

PubMed

Markert, Sven; Joeris, Klaus

2017-01-01

We developed an automated microtiter plate (MTP)-based system for suspension cell culture to meet the increased demands for miniaturized high throughput applications in biopharmaceutical process development. The generic system is based on off-the-shelf commercial laboratory automation equipment and is able to utilize MTPs of different configurations (6-24 wells per plate) in orbital shaken mode. The shaking conditions were optimized by Computational Fluid Dynamics simulations. The fully automated system handles plate transport, seeding and feeding of cells, daily sampling, and preparation of analytical assays. The integration of all required analytical instrumentation into the system enables a hands-off operation which prevents bottlenecks in sample processing. The modular set-up makes the system flexible and adaptable for a continuous extension of analytical parameters and add-on components. The system proved suitable as screening tool for process development by verifying the comparability of results for the MTP-based system and bioreactors regarding profiles of viable cell density, lactate, and product concentration of CHO cell lines. These studies confirmed that 6 well MTPs as well as 24 deepwell MTPs were predictive for a scale up to a 1000 L stirred tank reactor (scale factor 1:200,000). Applying the established cell culture system for automated media blend screening in late stage development, a 22% increase in product yield was achieved in comparison to the reference process. The predicted product increase was subsequently confirmed in 2 L bioreactors. Thus, we demonstrated the feasibility of the automated MTP-based cell culture system for enhanced screening and optimization applications in process development and identified further application areas such as process robustness. The system offers a great potential to accelerate time-to-market for new biopharmaceuticals. Biotechnol. Bioeng. 2017;114: 113-121. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches

PubMed Central

Singh, Pritika; Guleri, Rupam; Angurala, Amrita; Kaur, Kuldeep; Kaur, Kulwinder; Kaul, Sunil C.; Wadhwa, Renu

2017-01-01

Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field. PMID:28299323

Development of a new culture system for human lymphokine-activated killer cells: comparison with a conventional static culture method.

PubMed

Murata, M; Yano, T; Yoshino, I; Togami, M; Sogabe, M; Yasumoto, K; Sugimachi, K; Kimura, G; Nomoto, K

1991-10-01

We recently developed a new culture system based on dialysis perfusion (designated JCC-device) for the generation and expansion of human lymphokine-activated killer (LAK) cells (Murata et al., 1990). More recently we have scaled up the volume of the culture vessel of the JCC-device from 100 ml to 400 ml for clinical use. In the present study, using this new 400 ml JCC-device, we cultured human lymph node lymphocytes (LNL) obtained from 8 surgical patients with primary lung cancer, and investigated the cellular characteristics in comparison with a conventional batchwise culture system using tissue culture dishes. With the JCC-device, the cell density reached a maximum 2.7 x 10(7) cells/ml with greater than 90% viability by the appropriate exchange of perfusion medium and by making additions at the appropriate intervals for recombinant interleukin-2 (rIL-2). The expansion fold of LNL with the JCC-device, ranging 6.6- to 19.2-fold (mean 13.8-fold), was not significantly different from that in dish cultures. There was no marked difference in cell surface phenotypes between the two culture systems in 7 out of 8 cases. As for LAK activity of LNL, the JCC culture was either superior or equal in 4 out of 8 cases, but inferior in the other 4 cases to the conventional dish cultures. In the latter cases, the usage of serum for the JCC culture was limited, which might have resulted in the low LAK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

NASA Technical Reports Server (NTRS)

Tixador, R.

1992-01-01

The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.

PubMed

Hasselbach, Laura A; Irtenkauf, Susan M; Lemke, Nancy W; Nelson, Kevin K; Berezovsky, Artem D; Carlton, Enoch T; Transou, Andrea D; Mikkelsen, Tom; deCarvalho, Ana C

2014-01-07

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

PubMed Central

Hasselbach, Laura A.; Irtenkauf, Susan M.; Lemke, Nancy W.; Nelson, Kevin K.; Berezovsky, Artem D.; Carlton, Enoch T.; Transou, Andrea D.; Mikkelsen, Tom; deCarvalho, Ana C.

2014-01-01

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies. PMID:24429465

Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Hui; Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi; Pan, Wei-Kang

A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8more » was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin{sup +} cells decreased whilst the percentage of GFAP{sup +} cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. - Highlights: • Differences were identified between primary and daughter ENCCs. • Daughter ENCCs had reduced proliferation, migration and differentiation. • Daughter ENCCs also had increased apoptosis. • These altered characteristics warrant further investigation.« less

Microfluidics‐based 3D cell culture models: Utility in novel drug discovery and delivery research

PubMed Central

Gupta, Nilesh; Liu, Jeffrey R.; Patel, Brijeshkumar; Solomon, Deepak E.; Vaidya, Bhuvaneshwar

2016-01-01

Abstract The implementation of microfluidic devices within life sciences has furthered the possibilities of both academic and industrial applications such as rapid genome sequencing, predictive drug studies, and single cell manipulation. In contrast to the preferred two‐dimensional cell‐based screening, three‐dimensional (3D) systems have more in vivo relevance as well as ability to perform as a predictive tool for the success or failure of a drug screening campaign. 3D cell culture has shown an adaptive response to the recent advancements in microfluidic technologies which has allowed better control over spheroid sizes and subsequent drug screening studies. In this review, we highlight the most significant developments in the field of microfluidic 3D culture over the past half‐decade with a special focus on their benefits and challenges down the lane. With the newer technologies emerging, implementation of microfluidic 3D culture systems into the drug discovery pipeline is right around the bend. PMID:29313007

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation.

PubMed

Powers, Daryl E; Millman, Jeffrey R; Bonner-Weir, Susan; Rappel, Michael J; Colton, Clark K

2010-01-01

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO(2gas)). Because the cells are consuming oxygen supplied by diffusion, a difference between pO(2gas) and that experienced by the cells (pO(2cell)) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen-permeable silicone rubber membranes can be used to control pO(2cell) during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene-propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte-like increases with decreasing pO(2) only when using oxygen-permeable silicone membrane-based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO(2cell) being equal to pO(2gas) at the tissue-membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO(2) distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO(2cell), and these results suggest they are a valuable tool for investigating pO(2) effects in many applications, such as stem cell differentiation. Copyright 2009 American Institute of Chemical Engineers

Periodic harvesting of embryonic stem cells from a hollow-fiber membrane based four-compartment bioreactor.

PubMed

Knöspel, Fanny; Freyer, Nora; Stecklum, Maria; Gerlach, Jörg C; Zeilinger, Katrin

2016-01-01

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale-up of stem cell culture is necessary. Bioreactors for dynamic three-dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow-fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10(6) mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10(6) mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four-compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications.

PubMed

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-09-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

PubMed Central

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.

PubMed

Lin, Youshan Melissa; Lim, Jessica Fang Yan; Lee, Jialing; Choolani, Mahesh; Chan, Jerry Kok Yen; Reuveny, Shaul; Oh, Steve Kah Weng

2016-06-01

Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL, were investigated and compared in both conditions. BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.

PubMed

Kobayashi, Takehito; Yagi, Yusuke; Nakamura, Takahiro

2016-01-01

The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.

Growth, metabolic activity, and productivity of immobilized and freely suspended CHO cells in perfusion culture.

PubMed

Hilal-Alnaqbi, Ali; Hu, Alan Y C; Zhang, Zhibing; Al-Rubeai, Mohamed

2013-01-01

Chinese hamster ovary (CHO) cells producing β-galactosidase (β-gal) were successfully cultured on silicone-based porous microcarriers (ImmobaSil FS) in a 1 L stirred-tank perfusion bioreactor. We studied the growth, metabolism, and productivity of free and immobilized cells to understand cellular activity in immobilized conditions. CHO cells attached to ImmobaSil FS significantly better than to other microcarriers. Scanning electron microscope images showed that the CHO cells thoroughly colonized the porous surfaces of the ImmobaSil FS, exhibiting a spherical morphology with microvilli that extended to anchorage cells on the silicone surface. In perfusion culture, the concentration of the attached cells reached 8 × 10(8) cells/mL of carrier, whereas those that remained freely suspended reached 2 × 10(7) cells/mL medium. The β-gal concentration reached more than 5 unit/mL in perfusion culture, more than fivefold that of batch culture. The maximum concentration per microcarrier was proportional to the initial cell density. The specific growth rate, the specific β-gal production rate, the percentage of S phase, and the oxygen uptake rate were all relatively lower for immobilized cells than freely suspended cells in the same bioreactor, indicating that not only do cells survive and grow to a greater extent in a free suspension state, but they are also metabolically more active than viable cells inside the pores of the microcarriers. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Evaluation of a Multi-Parameter Sensor for Automated, Continuous Cell Culture Monitoring in Bioreactors

NASA Technical Reports Server (NTRS)

Pappas, D.; Jeevarajan, A.; Anderson, M. M.

2004-01-01

Compact and automated sensors are desired for assessing the health of cell cultures in biotechnology experiments in microgravity. Measurement of cell culture medium allows for the optirn.jzation of culture conditions on orbit to maximize cell growth and minimize unnecessary exchange of medium. While several discrete sensors exist to measure culture health, a multi-parameter sensor would simplify the experimental apparatus. One such sensor, the Paratrend 7, consists of three optical fibers for measuring pH, dissolved oxygen (p02), dissolved carbon dioxide (pC02) , and a thermocouple to measure temperature. The sensor bundle was designed for intra-arterial placement in clinical patients, and potentially can be used in NASA's Space Shuttle and International Space Station biotechnology program bioreactors. Methods: A Paratrend 7 sensor was placed at the outlet of a rotating-wall perfused vessel bioreactor system inoculated with BHK-21 (baby hamster kidney) cells. Cell culture medium (GTSF-2, composed of 40% minimum essential medium, 60% L-15 Leibovitz medium) was manually measured using a bench top blood gas analyzer (BGA, Ciba-Corning). Results: A Paratrend 7 sensor was used over a long-term (>120 day) cell culture experiment. The sensor was able to track changes in cell medium pH, p02, and pC02 due to the consumption of nutrients by the BHK-21. When compared to manually obtained BGA measurements, the sensor had good agreement for pH, p02, and pC02 with bias [and precision] of 0.02 [0.15], 1 mm Hg [18 mm Hg], and -4.0 mm Hg [8.0 mm Hg] respectively. The Paratrend oxygen sensor was recalibrated (offset) periodically due to drift. The bias for the raw (no offset or recalibration) oxygen measurements was 42 mm Hg [38 mm Hg]. The measured response (rise) time of the sensor was 20 +/- 4s for pH, 81 +/- 53s for pC02, 51 +/- 20s for p02. For long-term cell culture measurements, these response times are more than adequate. Based on these findings , the Paratrend sensor could offer automated, continuous monitoring of cell cultures with a temporal resolution of 1 minute, which is not attainable by sampling via handheld blood analyzer (i-STAT). Conclusion: The resulting bias and precision found in these cell culture-based studies is comparable to Paratrend sensor clinical results. Although the large error in p02 measurements (+/-18 mm Hg) may be acceptable for clinical applications, where Paratrend values are periodically adjusted to a BGA measurement, the O2 sensor in this bundle may not be reliable enough for the single-calibration requirement of sensors used in NASA's bioreactors. The pH and pC02 sensors in the bundle are reliable and stable over the measurement period, and can be used without recalibration to measure cell cultures in rn.jcrogravity biotechnology experiments. Future work will test additional Paratrend sensors to provide statistical assessment of sensor performance.

Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

PubMed Central

Shakibaei, Mehdi; Buhrmann, Constanze; Kraehe, Patricia; Shayan, Parviz; Lueders, Cora; Goel, Ajay

2014-01-01

Objective Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. Methods High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Results Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Conclusion Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract). PMID:24404205

Curcumin chemosensitizes 5-fluorouracil resistant MMR-deficient human colon cancer cells in high density cultures.

PubMed

Shakibaei, Mehdi; Buhrmann, Constanze; Kraehe, Patricia; Shayan, Parviz; Lueders, Cora; Goel, Ajay

2014-01-01

Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50-60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).

Pluripotency of adult stem cells derived from human and rat pancreas

NASA Astrophysics Data System (ADS)

Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

A Rotating Bioreactor for Scalable Culture and Differentiation of Respiratory Epithelium

PubMed Central

Raredon, Micha Sam Brickman; Ghaedi, Mahboobe; Calle, Elizabeth A.; Niklason, Laura E.

2015-01-01

Respiratory epithelium is difficult to grow in vitro, as it requires a well-maintained polarizing air–liquid interface (ALI) to maintain differentiation. Traditional methods rely on permeable membrane culture inserts, which are difficult to work with and are ill-suited for the production of large numbers of cells, such as the quantities required for cell-based clinical therapies. Herein, we investigate an alternative form of culture in which the cells are placed on a porous substrate that is continuously rolled, such that the monolayer of cells is alternately submerged in media or apically exposed to air. Our prototype bioreactor is reliable for up to 21 days of continuous culture and is designed for scale-up for large-scale cell culture with continuous medium and gas exchange. Normal human bronchial epithelial (NHBE) cells were cultured on an absorbent substrate in the reactor for periods of 7, 14, and 21 days and were compared to static controls that were submerged in media. Quantification by immunohistochemistry and quantitative PCR of markers specific to differentiated respiratory epithelium indicated increased cilia, mucous production, and tight junction formation in the rolled cultures, compared to static. Together with scanning electron microscopy and paraffin histology, the data indicate that the intermittent ALI provided by the rolling bioreactor promotes a polarized epithelial phenotype over a period of 21 days. PMID:26858899

[Experimental study on co-culture of salivary adenoid cystic carcinoma cells and ganglia].

PubMed

Gu, Ling; Bu, Rong-fa; Wang, Dong-sheng; E, Ling-ling; Zhu, Guo-xiong

2012-01-01

To construct the co-culture models of salivarya denoid cystic carcinoma (SACC) cells and dorsal root ganglia (DRG) of chickens and investigate the promotive effects of SACC on neural tissue. Glass-base culture dish was adopted to construct co-culture model of SACC-83 cells and DRG. SACC-83 cells were seeded in the medium pore with DRG around them. Outgrowth of neuronal processes was observed. Then DRG was cultured in the conditioned medium of SACC-83, with the groups of conditioned medium of MC3T3-E1 and HGF, the group of cell lysis buffer, the groups of serum-free medium and serum-plus medium as the controls. Outgrowth of neuronal processes was also recorded and compared with control groups. In the co-culture model of tumor and neuronal tissue, SACC-83 cells produced a suitable microenvironment in which neuronal processes remarkably grow. Neuronal processes of most DRG displayed growth tendency toward SACC. The group of conditioned medium from SACC-83 manifested obvious promotive effects on DRG. Co-culture model of tumor and neuronal tissue was successfully constructed, with which the promotive effects of tumor on outgrowth of neuronal processes could be observed. So hypothesized that SACC could secrete some neurotrophic factors to guide peripheral nerves gemmating and to trigger the cascade of the neural invasion in succession.

Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Danker, Timm; Wagner, Silvia; Hennenlotter, Joerg; Guenther, Elke; Obermayr, Florian; Stenzl, Arnulf; Koenigsrainer, Alfred; Skutella, Thomas; Just, Lothar

2009-12-01

Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

PubMed

Liverani, Chiara; La Manna, Federico; Groenewoud, Arwin; Mercatali, Laura; Van Der Pluijm, Gabri; Pieri, Federica; Cavaliere, Davide; De Vita, Alessandro; Spadazzi, Chiara; Miserocchi, Giacomo; Bongiovanni, Alberto; Recine, Federica; Riva, Nada; Amadori, Dino; Tasciotti, Ennio; Snaar-Jagalska, Ewa; Ibrahim, Toni

2017-02-15

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays. © 2016. Published by The Company of Biologists Ltd.

Raman Spectroscopic Analyses of Jaw Periosteal Cell Mineralization

PubMed Central

Brauchle, Eva; Carvajal Berrio, Daniel; Rieger, Melanie; Schenke-Layland, Katja; Reinert, Siegmar

2017-01-01

To achieve safer patient treatments, serum-free cell culture conditions have to be established for cell therapies. In previous studies, we demonstrated that serum-free culture favored the proliferation of MSCA-1+ osteoprogenitors derived from the jaw periosteum. In this study, the in vitro formation of bone-specific matrix by MSCA-1+ jaw periosteal cells (JPCs, 3 donors) was assessed and compared under serum-free and serum-containing media conditions using the marker-free Raman spectroscopy. Based on a standard fluorescence assay, JPCs from one patient were not able to mineralize under serum-containing culture conditions, whereas the other cells showed similar mineralization levels under both conditions. Raman spectra from mineralizing MSCA-1+ JPCs revealed higher levels of hydroxyapatite formation and higher mineral to matrix ratios under serum-free culture conditions. Higher carbonate to phosphate ratios and higher crystallinity in JPCs cultured under serum-containing conditions indicated immature bone formation. Due to reduced collagen production under serum-free conditions, we obtained significant differences in collagen maturity and proline to hydroxyproline ratios compared to serum-free conditions. We conclude that Raman spectroscopy is a useful tool for the assessment and noninvasive monitoring of in vitro mineralization of osteoprogenitor cells. Further studies should extend this knowledge and improve JPC mineralization by optimizing culture conditions. PMID:28232849

Novel Surface-Enhanced Raman Scattering-based Assays for Ultra-sensitive Detection of Human Pluripotent Stem Cells

PubMed Central

Han, Jingjia; Qian, Ximei; Wu, Qingling; Jha, Rajneesh; Duan, Jinshuai; Yang, Zhou; Maher, Kevin O.; Nie, Shuming; Xu, Chunhui

2017-01-01

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 106 cells, a sensitivity (0.0001%) which was ~2,000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5+ and TRA-1-60+ cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications. PMID:27509304

Selective Expansion of Skeletal Muscle Stem Cells from Bulk Muscle Cells in Soft Three‐Dimensional Fibrin Gel

PubMed Central

Zhu, Pei; Zhou, Yalu; Wu, Furen; Hong, Yuanfan; Wang, Xin; Shekhawat, Gajendra; Mosenson, Jeffrey

2017-01-01

Abstract Muscle stem cells (MuSCs) exhibit robust myogenic potential in vivo, thus providing a promising curative treatment for muscle disorders. Ex vivo expansion of adult MuSCs is highly desired to achieve a therapeutic cell dose because of their scarcity in limited muscle biopsies. Sorting of pure MuSCs is generally required for all the current culture systems. Here we developed a soft three‐dimensional (3D) salmon fibrin gel culture system that can selectively expand mouse MuSCs from bulk skeletal muscle preparations without cell sorting and faithfully maintain their regenerative capacity in culture. Our study established a novel platform for convenient ex vivo expansion of MuSCs, thus greatly advancing stem cell‐based therapies for various muscle disorders. Stem Cells Translational Medicine 2017;6:1412–1423 PMID:28244269

Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue.

PubMed

Juhl, Morten; Tratwal, Josefine; Follin, Bjarke; Søndergaard, Rebekka H; Kirchhoff, Maria; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana

2016-01-01

The utility of mesenchymal stromal cells (MSCs) in therapeutic applications for regenerative medicine has gained much attention. Clinical translation of MSC-based approaches requires in vitro culture-expansion to achieve a sufficient number of cells. The ideal cell culture medium should be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three different commercially available hPL fulfilling good manufacturing practice criteria for clinical use. BMSCs and ASCs cultured in Minimum Essential Medium Eagle-alpha supplemented with 5% PLT-Max (Mill Creek), Stemulate™ PL-S and Stemulate™ PL-SP (COOK General Biotechnology) were compared to standard culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS medium. In general, the immunophenotype of both BMSCs and ASCs fulfilled International Society for Cellular Therapy (ISCT) criteria after hPL media expansion. Comparative genomic hybridization measurements demonstrated no unbalanced chromosomal rearrangements for BMSCs or ASCs cultured in hPL media or FBS medium. The BMSCs and ASCs could differentiate into osteogenic, adipogenic, or chondrogenic lineages in all four culture conditions. All three clinically approved commercial human platelet lysates accelerated proliferation of BMSCs and ASCs and the cells meet the ISCT mesenchymal phenotypic requirements without exhibiting chromosomal aberrations.

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

NASA Technical Reports Server (NTRS)

Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

1998-01-01

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

Traditional Chinese medicine herbal mixture LQ arrests FUCCI-expressing HeLa cells in G₀/G₁ phase in 2D plastic, 2.5D Matrigel, and 3D Gelfoam culture visualized with FUCCI imaging.

PubMed

Zhang, Lei; Wu, Chengyu; Bouvet, Michael; Yano, Shuya; Hoffman, Robert M

2015-03-10

We used the fluorescence ubiquitination-based cell cycle indicator (FUCCI) to monitor cell cycle arrest after treatment of FUCCI-expressing HeLa cells (FUCCI-HeLa) with a traditional Chinese medicine (TCM) herbal mixture LQ, previously shown to have anti-tumor and anti-metastatic activity in mouse models. Paclitaxel was used as the positive control. In 2D monolayer culture, the untreated control had approximately 45% of the cells in S/G₂/M phase. In contrast, the LQ-treated cells (9 mg/ml) were mostly in the G₀/G₁ (>90%) after 72 hours. After treatment with paclitaxel (0.01 μm), for 72 hours, 95% of the cells were in S/G₂/M. In 2.5D Matrigel culture, the colonies in the untreated control group had 40% of the cells in S/G₂/M. LQ arrested the cells in G₀/G₁ after 72 hours. Paclitaxel arrested almost all the cells in S/G₂/M after 72 hours. In 3D Gelfoam culture, the untreated control culture had approximately 45% of cells in G₂/M. In contrast, the LQ-treated cells were mostly in G₀/G₁ phase (>80%) after 72 hours treatment. Paclitaxel resulted in 90% of the cells arrested in S/G₂/M after 72 hours. The present report suggests the non-toxic LQ has potential to maintain cancers in a quiescent state for long periods of time.

Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

PubMed

Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

2012-12-01

The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

Electrolytic Valving Isolation for Cell Co-Culture Microenvironment with Controlled Cell Pairing Ratios

PubMed Central

Chen, Yu-Chih; Ingram, Patrick; Yoon, Euisik

2016-01-01

Cancer-stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we present a cell-cell interaction microfluidic platform that can accurately control co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We verified that electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we performed cell-cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell-cell interaction assays was successfully performed showing that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells. PMID:25118341

Thermogelling 3D Systems towards Stem Cell-Based Tissue Regeneration Therapies.

PubMed

Wang, Xiaoyuan; Young, David James; Wu, Yun-Long; Loh, Xian Jun

2018-03-02

Stem cell culturing and differentiation is a very important research direction for tissue engineering. Thermogels are well suited for encapsulating cells because of their non-biotoxic nature and mild sol-gel transition as temperature increases. In particular, thermogels provide a 3D growth environment for stem cell growth, which is more similar to the extracellular matrix than flat substrates, so thermogels as a medium can overcome many of the cell abnormalities caused by 2D cell growth. In this review, we summarize the applications of thermogels in cell and stem cell culture in recent years. We also elaborate on the methods to induce stem cell differentiation by using thermogel-based 3D scaffolds. In particular, thermogels, encapsulating specific differentiation-inducing factor and having specific structures and moduli, can induce the differentiation into the desired tissue cells. Three dimensional thermogel scaffolds that control the growth and differentiation of cells will undoubtedly have a bright future in regenerative medicine.

Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.

PubMed

Bonakdar, Shahin; Mahmoudi, Morteza; Montazeri, Leila; Taghipoor, Mojtaba; Bertsch, Arnaud; Shokrgozar, Mohammad Ali; Sharifi, Shahriar; Majidi, Mohammad; Mashinchian, Omid; Hamrang Sekachaei, Mohammad; Zolfaghari, Pegah; Renaud, Philippe

2016-06-08

Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.

Gelatin-based microcarriers as embryonic stem cell delivery system in bone tissue engineering: an in-vitro study.

PubMed

Tielens, S; Declercq, H; Gorski, T; Lippens, E; Schacht, E; Cornelissen, M

2007-03-01

Mouse embryonic stem cells were cultured on commercially available biodegradable macroporous microcarriers. A culture period of 1-2 weeks was needed to colonize the microcarriers. Embryonic stem cells retained their pluripotency for up to 14 days when cultured in medium supplemented with leukemia inhibitory factor. Replacing this medium by differentiation medium for 2 weeks initiated osteogenic differentiation. Encapsulation of the cell-loaded microcarriers in photopolymerizable polymers (methacrylate-endcapped poly-D,L-lactide-co-caprolactone), triacetin/hydroxyethylmethacrylate (HEMA) as solvent and with/without gelatin as porogen, resulted in a homogeneous distribution of the microcarriers in the polymer. As observed by transmission electron microscopy, viability of the cells was optimal when gelatin was omitted and when using triacetin instead of HEMA.

Magnetic-Activated Cell Sorting for the Fast and Efficient Separation of Human and Rodent Schwann Cells from Mixed Cell Populations.

PubMed

Ravelo, Kristine M; Andersen, Natalia D; Monje, Paula V

2018-01-01

To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75 NGFR , O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.

Adipose tissue-organotypic culture system as a promising model for studying adipose tissue biology and regeneration

PubMed Central

Uchihashi, Kazuyoshi; Aoki, Shigehisa; Sonoda, Emiko; Yamasaki, Fumio; Piao, Meihua; Ootani, Akifumi; Yonemitsu, Nobuhisa; Sugihara, Hajime

2009-01-01

Adipose tissue consists of mature adipocytes, preadipocytes and mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. We have recently developed “adipose tissue-organotypic culture system” that maintains unilocular structure, proliferative ability and functions of mature adipocytes for a long term, using three-dimensional collagen gel culture of the tissue fragments. In this system, both preadipocytes and MSCs regenerate actively at the peripheral zone of the fragments. Our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome. Thus, it seems to be a promising model for investigating adipose tissue biology and regeneration. In this article, we introduce adipose tissue-organotypic culture, and propose two theories regarding the mechanism of tissue regeneration that occurs specifically at peripheral zone of tissue fragments in vitro. PMID:19794899

Impact of environmental factors on the culturability and viability of Listeria monocytogenes under conditions encountered in food processing plants.

PubMed

Overney, Anaïs; Jacques-André-Coquin, Joséphine; Ng, Patricia; Carpentier, Brigitte; Guillier, Laurent; Firmesse, Olivier

2017-03-06

The ability of Listeria monocytogenes to adhere to and persist on surfaces for months or even years may be responsible for its transmission from contaminated surfaces to food products. Hence the necessity to find effective means to prevent the establishment of L. monocytogenes in food processing environments. The aim of this study was to assess, through a fractional experimental design, the environmental factors that could affect the survival of L. monocytogenes cells on surfaces to thereby prevent the persistence of this pathogen in conditions mimicking those encountered in food processing plants: culture with smoked salmon juice or meat exudate, use of two materials with different hygiene status, biofilm of L. monocytogenes in pure-culture or dual-culture with a Pseudomonas fluorescens strain, application of a drying step after cleaning and disinfection (C&D) and comparison of two strains of L. monocytogenes. Bacterial survival was assessed by culture, qPCR to quantify total cells, and propidium monoazide coupled with qPCR to quantify viable cells and highlight viable but non-culturable (VBNC) cells. Our results showed that failure to apply C&D causes cell persistence on surfaces. Moreover, the sanitation procedure leads only to a loss of culturability and appearance of VBNC populations. However, an additional daily drying step after C&D optimises the effectiveness of these procedures to reduce culturable populations. Our results reinforce the importance to use molecular tools to monitor viable pathogens in food processing plants to avoid underestimating the amounts of cells using only methods based on cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors.

PubMed

Khurshid, Madiha; Mulet-Sierra, Aillette; Adesida, Adetola; Sen, Arindom

2018-03-01

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair. Copyright © 2017 John Wiley & Sons, Ltd.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

PubMed

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

PubMed

Fu, Jilagamazhi; Sharma, Umesh; Sparling, Richard; Cicek, Nazim; Levin, David B

2014-07-01

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

Three-dimensional hydrogel cell culture systems for modeling neural tissue

NASA Astrophysics Data System (ADS)

Frampton, John

Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was designed for use as a tool to predict the transport and processing that occurs prior to drug uptake in the central nervous system (CNS), and to predict BBB permeability. Electrochemical techniques and immunohistochemistry were used to validate this model and provide detailed information about cellular organization and function. Electrochemical impedance spectroscopy (EIS) provided evidence that endothelial cells cultured in the presence of astrocytes formed tight junctions capable of occluding the flow of electrical current. In a second series of experiments, a microglia-astrocyte co-culture system was developed to assess the effects of glial cells on electrode impedance recorded from neural prosthetic devices in vitro. Impedance measurements were compared with confocal images to determine the effects of glial cell density and cell type on electrode performance. The results indicate that EIS data can be used to model components of the reactive cell responses in brain tissue, and that impedance measurements recorded in vitro can be compared to measurements recorded in vivo. Taken together, these results demonstrate that alginate hydrogels can be used for the creation of 3-D neural cell scaffolds, and that such cell scaffolds can be used to model a variety of three-dimensional neural tissues in vitro, that cannot be studied in 2-D cultures.

Managing evaporation for more robust microscale assays. Part 2. Characterization of convection and diffusion for cell biology.

PubMed

Berthier, Erwin; Warrick, Jay; Yu, Hongmeiy; Beebe, David J

2008-06-01

Cell based microassays allow the screening of a multitude of culture conditions in parallel, which can be used for various applications from drug screening to fundamental cell biology research. Tubeless microfluidic devices based on passive pumping are a step towards accessible high throughput microassays, however they are vulnerable to evaporation. In addition to volume loss, evaporation can lead to the generation of small flows. Here, we focus on issues of convection and diffusion for cell culture in microchannels and particularly the transport of soluble factors secreted by cells. We find that even for humidity levels as high as 95%, convection in a passive pumping channel can significantly alter distributions of these factors and that appropriate system design can prevent convection.

Assay based on electrical impedance spectroscopy to discriminate between normal and cancerous mammalian cells

NASA Astrophysics Data System (ADS)

Giana, Fabián Eduardo; Bonetto, Fabián José; Bellotti, Mariela Inés

2018-03-01

In this work we present an assay to discriminate between normal and cancerous cells. The method is based on the measurement of electrical impedance spectra of in vitro cell cultures. We developed a protocol consisting on four consecutive measurement phases, each of them designed to obtain different information about the cell cultures. Through the analysis of the measured data, 26 characteristic features were obtained for both cell types. From the complete set of features, we selected the most relevant in terms of their discriminant capacity by means of conventional statistical tests. A linear discriminant analysis was then carried out on the selected features, allowing the classification of the samples in normal or cancerous with 4.5% of false positives and no false negatives.

Assay based on electrical impedance spectroscopy to discriminate between normal and cancerous mammalian cells.

PubMed

Giana, Fabián Eduardo; Bonetto, Fabián José; Bellotti, Mariela Inés

2018-03-01

In this work we present an assay to discriminate between normal and cancerous cells. The method is based on the measurement of electrical impedance spectra of in vitro cell cultures. We developed a protocol consisting on four consecutive measurement phases, each of them designed to obtain different information about the cell cultures. Through the analysis of the measured data, 26 characteristic features were obtained for both cell types. From the complete set of features, we selected the most relevant in terms of their discriminant capacity by means of conventional statistical tests. A linear discriminant analysis was then carried out on the selected features, allowing the classification of the samples in normal or cancerous with 4.5% of false positives and no false negatives.

Fabrication of cell-benign inverse opal hydrogels for three-dimensional cell culture.

PubMed

Im, Pilseon; Ji, Dong Hwan; Kim, Min Kyung; Kim, Jaeyun

2017-05-15

Inverse opal hydrogels (IOHs) for cell culture were fabricated and optimized using calcium-crosslinked alginate microbeads as sacrificial template and gelatin as a matrix. In contrast to traditional three-dimensional (3D) scaffolds, the gelatin IOHs allowed the utilization of both the macropore surface and inner matrix for cell co-culture. In order to remove templates efficiently for the construction of 3D interconnected macropores and to maintain high cell viability during the template removal process using EDTA solution, various factors in fabrication, including alginate viscosity, alginate concentration, alginate microbeads size, crosslinking calcium concentration, and gelatin network density were investigated. Low viscosity alginate, lower crosslinking calcium ion concentration, and lower concentration of alginate and gelatin were found to obtain high viability of cells encapsulated in the gelatin matrix after removal of the alginate template by EDTA treatment by allowing rapid dissociation and diffusion of alginate polymers. Based on the optimized fabrication conditions, gelatin IOHs showed good potential as a cell co-culture system, applicable to tissue engineering and cancer research. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction

PubMed Central

Mostofi, Sepideh; Bonyadi Rad, Ehsan; Wiltsche, Helmar; Fasching, Ulrike; Szakacs, Gabor; Ramskogler, Claudia; Srinivasaiah, Sriveena; Ueçal, Muammer; Willumeit, Regine; Weinberg, Annelie-Martina; Schaefer, Ute

2016-01-01

This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for in vivo orthopedic applications. PMID:27459513

Induction of differentiation of human embryonic stem cells into functional hair-cell-like cells in the absence of stromal cells.

PubMed

Ding, Jie; Tang, Zihua; Chen, Jiarong; Shi, Haosong; Chen, Jianling; Wang, Cuicui; Zhang, Cui; Li, Liang; Chen, Ping; Wang, Jinfu

2016-12-01

Sensorineural hearing loss and vestibular dysfunction have become the most common forms of sensory defects. Stem cell-based therapeutic strategies for curing hearing loss are being developed. Several attempts to develop hair cells by using chicken utricle stromal cells as feeder cells have resulted in phenotypic conversion of stem cells into inner ear hair-cell-like cells. Here, we induced the differentiation of human embryonic stem cells (hESCs) into otic epithelial progenitors (OEPs), and further induced the differentiation of OEPs into hair-cell-like cells using different substrates. Our results showed that OEPs cultured on the chicken utricle stromal cells with the induction medium could differentiate into hair-cell-like cells with stereociliary bundles. Co-culture with stromal cells, however, may be problematic for subsequent examination of the induced hair-cell-like cells. In order to avoid the interference from stromal cells, we cultured OEPs on laminin with different induction media and examined the effects of the induction medium on the differentiation potentials of OEPs into hair-cell-like cells. The results revealed that the culture of OEPs on laminin with the conditioned medium from chicken utricle stromal cells supplemented with EGF and all-trans retinoic acid (RA) could promote the organization of cells into epithelial clusters displaying hair-cell-like cells with stereociliary bundles. These cells also displayed the expected electrophysiological properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

PubMed

Amaral, Robson L F; Miranda, Mariza; Marcato, Priscyla D; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 10 4 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 10 4 cells/mL for the ULA method and 2.5 and 3.75 × 10 4 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC 50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC 50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

PubMed Central

Amaral, Robson L. F.; Miranda, Mariza; Marcato, Priscyla D.; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300–500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application. PMID:28878686

Application of a cell-once-through perfusion strategy for production of recombinant antibody from rCHO cells in a Centritech Lab II centrifuge system.

PubMed

Kim, Byoung Jin; Chang, Ho Nam; Oh, Duk Jae

2007-01-01

Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.

Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses

PubMed Central

Jazaeri Farsani, Seyed Mohammad; Deijs, Martin; Dijkman, Ronald; Molenkamp, Richard; Jeeninga, Rienk E; Ieven, Margareta; Goossens, Herman; van der Hoek, Lia

2015-01-01

Background Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses. Method Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air–liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique. Results Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively). Conclusion We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates. PMID:25482367

Open-dish incubator for live cell imaging with an inverted microscope.

PubMed

Heidemann, Steven R; Lamoureux, Phillip; Ngo, Kha; Reynolds, Matthew; Buxbaum, Robert E

2003-10-01

Here we describe the design and fabrication of an inexpensive cell culture incubator for the stage of an inverted light microscope for use in live cell imaging. This device maintains the temperature of the cell culture at 37 degrees C with great stability and, after reaching equilibrium, provides focal stability of an image for 20-25 min with oil-immersion lenses. We describe two versions of the incubator: one for use with standard 60-mm plastic culture dishes, and the other version for imaging of cells on glass coverslips. Either can be made for less than $400. Most components are widely available commercially, and it requires only simple wiring and 3 h to assemble. Although the device is generally useful for live cell imaging on an inverted microscope, it is particularly suitable for work in which instruments are introduced into the culture, such as electrophysiology or micromanipulation. The design is based on the principle that control performance is limited by the lag time between detection and response. The key element of the design is a heated, temperature-controlled aluminum ring serving as a mini-incubator surrounding the culture vessel. For this reason, we call our design a "ringcubator."

Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing.

PubMed

Koch, R J; Goode, R L; Simpson, G T

1997-04-01

The purpose of this study was to develop an in vitro serum-free keloid fibroblast model. Keloid formation remains a problem for every surgeon. Prior evaluations of fibroblast characteristics in vitro, especially those of growth factor measurement, have been confounded by the presence of serum-containing tissue culture media. The serum itself contains growth factors, yet has been a "necessary evil" to sustain cell growth. The design of this study is laboratory-based and uses keloid fibroblasts obtained from five patients undergoing facial (ear lobule) keloid removal in a university-affiliated clinic. Keloid fibroblasts were established in primary cell culture and then propagated in a serum-free environment. The main outcome measures included sustained keloid fibroblast growth and viability, which was comparable to serum-based models. The keloid fibroblast cell cultures exhibited logarithmic growth, sustained a high cellular viability, maintained a monolayer, and displayed contact inhibition. Demonstrating model consistency, there was no statistically significant difference between the mean cell counts of the five keloid fibroblast cell lines at each experimental time point. The in vitro growth of keloid fibroblasts in a serum-free model has not been done previous to this study. The results of this study indicate that the proliferative characteristics described are comparable to those of serum-based models. The described model will facilitate the evaluation of potential wound healing modulators, and cellular effects and collagen modifications of laser resurfacing techniques, and may serve as a harvest source for contaminant-free fibroblast autoimplants. Perhaps its greatest utility will be in the evaluation of endogenous and exogenous growth factors.

Cell Culture in Microgravity: Opening the Door to Space Cell Biology

NASA Technical Reports Server (NTRS)

Pellis, Neal R.; Dawson, David L. (Technical Monitor)

1999-01-01

Adaptational response of human cell populations to microgravity is investigated using simulation, short-term Shuttle experiments, and long-term microgravity. Simulation consists of a clinostatically-rotated cell culture system. The system is a horizontally-rotated cylinder completely filled with culture medium. Low speed rotation results in continuous-fall of the cells through the fluid medium. In this setting, cells: 1) aggregate, 2) propagate in three dimensions, 3) synthesize matrix, 4) differentiate, and 5) form sinusoids that facilitate mass transfer. Space cell culture is conducted in flight bioreactors and in static incubators. Cells grown in microgravity are: bovine cartilage, promyelocytic leukemia, kidney proximal tubule cells, adrenal medulla, breast and colon cancer, and endothelium. Cells were cultured in space to test specific hypotheses. Cartilage cells were used to determine structural differences in cartilage grown in space compared to ground-based bioreactors. Results from a 130-day experiment on Mir revealed that cartilage grown in space was substantially more compressible due to insufficient glycosaminoglycan in the matrix. Interestingly, earth-grown cartilage conformed better to the dimensions of the scaffolding material, while the Mir specimens were spherical. The other cell populations are currently being analyzed for cell surface properties, gene expression, and differentiation. Results suggest that some cells spontaneously differentiate in microgravity. Additionally, vast changes in gene expression may occur in response to microgravity. In conclusion, the transition to microgravity may constitute a physical perturbation in cells resulting in unique gene expressions, the consequences of which may be useful in tissue engineering, disease modeling, and space cell biology.

A cell stabilization factor for transport of experimental cell cultures to and from the International Space Station

NASA Astrophysics Data System (ADS)

Fattaey, Heideh K.; Consigli, Richard A.; Grenz, Ladonna; Johnson, Terry C.

2000-01-01

The requirement for long term storage of cell cultures previous to arrival on the International Space Station (ISS), as well as culture maintenance after the conduct of experiment in microgravity, necessitates inhibition of cell proliferation and metabolism pending return to earth-based laboratories. Transport of cells in a nonstabilized condition can lead to a loss of cell viability and/or a source of selection pressures for survival that can alter the overall cell population. We have isolated in our laboratory a reversible inhibitor of cell proliferation, a cell regulatory sialoglycopeptide (CeReS-18), that has the capability of stabilizing cells isolated from a wide phylogenetic range by arresting them in the G1 phase of the cell cycle. We show here that CeReS-18 is unusually stable and can be stored at ambient temperatures for weeks without a measurable loss in its biological activity. In addition we demonstrate that CeReS-18 is a superior cell-stabilizing agent as compared to other methods deployed for cell stabilization purposes, such as, decrease in the incubation temperature and serum down shifts. We also discovered that hybridoma cultures stabilized in their proliferative cycle by CeReS-18 produced 150%-300% more antibody per cell than that measured in the proliferating control cultures. The reversible inhibitory activity of CeReS-18, together with its unusual stability, as well as its wide target range lend themselves to use of this inhibitor as a cell stabilizing agent for cell transport to and storage on the ISS. .

Magnetically controllable 3D microtissues based on magnetic microcryogels.

PubMed

Liu, Wei; Li, Yaqian; Feng, Siyu; Ning, Jia; Wang, Jingyu; Gou, Maling; Chen, Huijun; Xu, Feng; Du, Yanan

2014-08-07

Microtissues on the scale of several hundred microns are a promising cell culture configuration resembling the functional tissue units in vivo. In contrast to conventional cell culture, handling of microtissues poses new challenges such as medium exchange, purification and maintenance of the microtissue integrity. Here, we developed magnetic microcryogels to assist microtissue formation with enhanced controllability and robustness. The magnetic microcryogels were fabricated on-chip by cryogelation and micro-molding which could endure extensive external forces such as fluidic shear stress during pipetting and syringe injection. The magnetically controllable microtissues were applied to constitute a novel separable 3D co-culture system realizing functional enhancement of the hepatic microtissues co-cultured with the stromal microtissues and easy purification of the hepatic microtissues for downstream drug testing. The magnetically controllable microtissues with pre-defined shapes were also applied as building blocks to accelerate the tissue assembly process under magnetic force for bottom-up tissue engineering. Finally, the magnetic microcryogels could be injected in vivo as cell delivery vehicles and tracked by MRI. The injectable magnetic microtissues maintained viability at the injection site indicating good retention and potential applications for cell therapy. The magnetic microcryogels are expected to significantly promote the microtissues as a promising cellular configuration for cell-based applications such as in drug testing, tissue engineering and regenerative therapy.

PCR-based detection of a rare linear DNA in cell culture.

PubMed

Saveliev, Sergei V.

2002-11-11

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10(7) or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

PCR-based detection of a rare linear DNA in cell culture

PubMed Central

2002-01-01

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials. PMID:12734566

Zinc oxide nanostructures as low-cost templates for neuronal circuit

NASA Astrophysics Data System (ADS)

Kritharidou, A.; Georgoussi, Z.; Tsamis, C.; Makarona, E.

2013-05-01

ZnO nanostructures were explored as templates for the development of topography-mediated neuronal cultures. Nanostructures of varying features were produced on 4" Si substrates via a rapid, facile and low-cost technique that allows the systematic investigation of nanotopographically-mediated formation of neuronal cultures. The developed ZnO-nanowire based templates were seeded with Neuro-2A mouse neuroblastoma cells and their viability over the course of 1 to 4 days was assessed. Our studies demonstrate that the ZnO-templates can support neuronal cell growth and proliferation suggesting that ZnO substrate can be used for the development of neuronal cell-based platform technologies.

Growing B Lymphocytes in a Three-Dimensional Culture System

NASA Technical Reports Server (NTRS)

Wu, J. H. David; Bottaro, Andrea

2010-01-01

A three-dimensional (3D) culture system for growing long-lived B lymphocytes has been invented. The capabilities afforded by the system can be expected to expand the range of options for immunological research and related activities, including testing of immunogenicity of vaccine candidates in vitro, generation of human monoclonal antibodies, and immunotherapy. Mature lymphocytes, which are the effectors of adaptive immune responses in vertebrates, are extremely susceptible to apoptotic death, and depend on continuous reception of survival-inducing stimulation (in the forms of cytokines, cell-to-cell contacts, and antigen receptor signaling) from the microenvironment. For this reason, efforts to develop systems for long-term culture of functional, non-transformed and non-activated mature lymphocytes have been unsuccessful until now. The bone-marrow microenvironment supports the growth and differentiation of many hematopoietic lineages, in addition to B-lymphocytes. Primary bone-marrow cell cultures designed to promote the development of specific cell types in vitro are highly desirable experimental systems, amenable to manipulation under controlled conditions. However, the dynamic and complex network of stromal cells and insoluble matrix proteins is disrupted in prior plate- and flask-based culture systems, wherein the microenvironments have a predominantly two-dimensional (2D) character. In 2D bone-marrow cultures, normal B-lymphoid cells become progressively skewed toward precursor B-cell populations that do not retain a normal immunophenotype, and such mature B-lymphocytes as those harvested from the spleen or lymph nodes do not survive beyond several days ex vivo in the absence of mitogenic stimulation. The present 3D culture system is a bioreactor that contains highly porous artificial scaffolding that supports the long-term culture of bone marrow, spleen, and lymph-node samples. In this system, unlike in 2D culture systems, B-cell subpopulations developing within 3D cultures that have been modified to foster lymphopoiesis retain an immunophenotype that closely recapitulates cells in fresh bone marrow harvests. The 3D culture system has been found to be capable of supporting long-lived (8 weeks) populations of B and T lymphocytes from peripheral lymphoid organs, in the absence of activation signals, to an extent not achievable by conventional culture techniques. Interestingly, it has been found that 3D-culture B cells display a phenotype that has characteristics of both B1a and B2 cells. These promising preliminary observations suggest that the 3D culture system could be used with success in the study of peripheral-B-lymphocyte biology and in the development of biotechnological techniques and processes.

Relative biological effectiveness (RBE) of alpha radiation in cultured porcine aortic endothelial cells.

PubMed

Thomas, Patricia; Tracy, Bliss; Ping, Tilly; Baweja, Anar; Wickstrom, Mark; Sidhu, Narinder; Hiebert, Linda

2007-03-01

Northern peoples can receive elevated radiation doses (1- 10 mSv/y) from transfer of polonium-210 (210Po) through the lichen-caribou-human food chain. Ingested 210Po is primarily blood-borne and thus many of its short range alpha particles irradiate the endothelial cells lining the blood vessels. The relative biological effectiveness (RBE) of alpha particles vs. x-rays was examined in porcine aortic endothelial cells as a surrogate for understanding what might happen to human endothelial cells in northern populations consuming traditional foods. Cultured porcine aortic endothelial cells were exposed to x-ray and 210Po alpha particle radiation. Alpha irradiation was applied to the cell cultures internally via the culture medium and externally, using thin-bottomed culture dishes. The results given here are based on the external irradiation method, which was found to be more reliable. Dose-response curves were compared for four lethal endpoints (cell viability, live cell fraction, release of lactate dehydrogenase [LDH] and clonogenic survival) to determine the relative biological effectiveness (RBE) of alpha radiation. The alpha RBE for porcine cells varied from 1.6-21, depending on the endpoint: 21.2+/-4.5 for cell viability, 12.9+/-2.7 for decrease in live cell number, 5.3+/-0.4 for LDH release to the medium but only 1.6 +/-0.1 for clonogenic survival. The low RBE of 1.6 was due to x-ray hypersensitivity of endothelial cells at low doses.

A Direct Cell Quenching Method for Cell-Culture Based Metabolomics

EPA Science Inventory

A crucial step in metabolomic analysis of cellular extracts is the cell quenching process. The conventional method first uses trypsin to detach cells from their growth surface. This inevitably changes the profile of cellular metabolites since the detachment of cells from the extr...

Laboratory production of human prolactin from CHO cells adapted to serum-free suspension culture.

PubMed

Arthuso, Fernanda Santos; Bartolini, Paolo; Soares, Carlos Roberto Jorge

2012-08-01

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23 kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40 days, to grow in suspension in serum-free medium. High cell densities of up to 4.0 × 10(6) cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30 days. Two harvesting strategies were set up, 50 or 100 % of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5-7 μg hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.

Development of nutraceutical formulations based on the mycelium of Pleurotus ostreatus and Agaricus bisporus.

PubMed

Cardoso, Rossana V C; Fernandes, Ângela; Oliveira, M Beatriz P P; Calhelha, Ricardo C; Barros, Lillian; Martins, Anabela; Ferreira, Isabel C F R

2017-06-21

The present work is aimed at developing nutraceutical formulations based on the mycelium of Agaricus bisporus and Pleurotus ostreatus, highlighting the potential of in vitro culture as a tool to improve the production of bioactive compounds, namely phenolic acids and ergosterol. The mycelia of both species were cultured in different solid and liquid media in order to compare the growth rate and yielded biomass. Fruiting bodies, mycelia and culture media were compared regarding the antioxidant activity, anti-inflammatory effects in RAW264.7 cells and cytotoxicity in human tumor cell lines and non-tumor porcine liver cells. P. ostreatus mycelia showed higher contents of ergosterol and phenolic compounds, and stronger antioxidant activity than the corresponding fruiting body. P. ostreatus and A. bisporus did not show anti-inflammatory activity, and P. ostreatus was the only one showing cytotoxicity in tumor cell lines. The results show that these mushrooms provide compounds with antioxidant and cytotoxic capacities, with variations among species.

Cell sheet engineering using the stromal vascular fraction of adipose tissue as a vascularization strategy.

PubMed

Costa, Marina; Cerqueira, Mariana T; Santos, Tírcia C; Sampaio-Marques, Belém; Ludovico, Paula; Marques, Alexandra P; Pirraco, Rogério P; Reis, Rui L

2017-06-01

Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditions for up to 8days in the absence of extrinsic growth factors. Immunocytochemistry against CD31 and CD146 revealed spontaneous organization in capillary-like structures, more complex after hypoxic conditioning. Inhibition of HIF-1α pathway hindered capillary-like structure formation in SVF cells cultured in hypoxia, suggesting a role of HIF-1α. Moreover, hypoxic SVF cells showed a trend for increased secretion of angiogenic factors, which was reflected in increased network formation by endothelial cells cultured on matrigel using that conditioned medium. In vivo implantation of SVF CS in a mouse hind limb ischemia model revealed that hypoxia-conditioned CS led to improved restoration of blood flow. Both in vitro and in vivo data suggest that SVF CS can be used as simple and cost-efficient tools to promote functional vascularization of TE constructs. Neovascularization after implantation is a major obstacle for producing clinically viable cell sheet-based tissue engineered constructs. Strategies using endothelial cells and extrinsic angiogenic growth factors are expensive and time consuming and may raise concerns of tumorigenicity. In this manuscript, we describe a simplified approach using angiogenic cell sheets fabricated from the stromal vascular fraction of adipose tissue. The strong angiogenic behavior of these cell sheets, achieved without the use of external growth factors, was further stimulated by low oxygen culture. When implanted in an in vivo model of hind limb ischemia, the angiogenic cell sheets contributed to blood flux recovery. These cell sheets can therefore be used as a straightforward tool to increase the neovascularization of cell sheet-based thick constructs. Copyright © 2017. Published by Elsevier Ltd.

Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors.

PubMed

Titmarsh, Drew; Hidalgo, Alejandro; Turner, Jennifer; Wolvetang, Ernst; Cooper-White, Justin

2011-12-01

Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However, since cell fate is crucially dependent on this microenvironment, it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free, chemically defined conditions, and further, whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this, we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number <1), cells are affected by apparent nutrient depletion and waste accumulation, evidenced by reduced cell expansion and altered morphology. At higher rates, cells are spontaneously washed out, and display morphological changes which may be indicative of early-stage differentiation. However, between these thresholds exists a narrow range of flowrates in which hESCs expand comparably to the equivalent static culture system, with regular morphology and maintenance of the pluripotency marker TG30 in >95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures, which may therefore provide a good first estimate of appropriate perfusion rates. Overall, we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days, a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture. Copyright © 2011 Crown in the right of Canada.

Induction of human antigen-specific suppressor factors in vitro.

PubMed Central

Kontiainen, S; Woody, J N; Rees, A; Feldmann, M

1981-01-01

Based on methods used for the in vitro induction of antigen-specific suppressor cells in the mouse, we have cultured Ficoll-Isopaque-separated human blood cells with high dose of antigen (100 microgram/ml) in Marbrook culture vessels for 4 days. The resulting cells, when further recultured for 24 hr with a low dose of antigen (1 microgram/ml), released into the supernatant material, termed 'suppressor factor', which inhibited, in an antigen-specific manner, the antibody response of mouse spleen cells in culture. The suppressor factor was analysed using immunoabsorbents, and was bound to and eluted from specific antigen, concanavalin A and lentil lectin, anti-human Ia antibodies, and anti-mouse suppressor factor antibodies, but was not bound to antibodies against human IgG. PMID:6169475

Cell separation and electrofusion in space

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Hofmann, G. A.

1990-01-01

In microgravity, free-fluid electrophoretic methods for separating living cells and proteins are improved significantly by the absence of gravity-driven phenomena. Cell fusion, culture, and other bioprocessing steps are being investigated to understand the limits of earth-based processing. A multistep space bioprocess is described that includes electrophoretic separation of human target cells, single-cell manipulations using receptor-specific antibodies, electrofusion to produce immortal hybridomas, gentle suspension culture, and monoclonal antibody recovery using continuous-flow electrophoresis or recirculating isoelectric focusing. Improvements in several key steps already have been demonstrated by space experiments, and others will be studied on Space Station Freedom.

EVALUATING VIRULENCE OF WATERBORNE AND CLINCIAL AEROMONAS ISOLATES USING GENE EXPRESSION AND MORTALITY IN NEONATAL MICE FOLLOWED BY ASSESSING CELL CULTURE'S ABILITY TO PREDICT VIRULENCE BASED ON TRANSCRIPTIONAL RESPONSE

EPA Science Inventory

The virulence of multiple Aeromonas spp. were assessed using two models, a neonatal mouse assay and a mouse intestinal cell culture. Transcriptional responses to both infection models were assessed using microarrays. After artificial infection with a variety of Aeromonas spp., ...

Mammalian cell culture process for monoclonal antibody production: nonlinear modelling and parameter estimation.

PubMed

Selişteanu, Dan; Șendrescu, Dorin; Georgeanu, Vlad; Roman, Monica

2015-01-01

Monoclonal antibodies (mAbs) are at present one of the fastest growing products of pharmaceutical industry, with widespread applications in biochemistry, biology, and medicine. The operation of mAbs production processes is predominantly based on empirical knowledge, the improvements being achieved by using trial-and-error experiments and precedent practices. The nonlinearity of these processes and the absence of suitable instrumentation require an enhanced modelling effort and modern kinetic parameter estimation strategies. The present work is dedicated to nonlinear dynamic modelling and parameter estimation for a mammalian cell culture process used for mAb production. By using a dynamical model of such kind of processes, an optimization-based technique for estimation of kinetic parameters in the model of mammalian cell culture process is developed. The estimation is achieved as a result of minimizing an error function by a particle swarm optimization (PSO) algorithm. The proposed estimation approach is analyzed in this work by using a particular model of mammalian cell culture, as a case study, but is generic for this class of bioprocesses. The presented case study shows that the proposed parameter estimation technique provides a more accurate simulation of the experimentally observed process behaviour than reported in previous studies.

Mammalian Cell Culture Process for Monoclonal Antibody Production: Nonlinear Modelling and Parameter Estimation

PubMed Central

Selişteanu, Dan; Șendrescu, Dorin; Georgeanu, Vlad

2015-01-01

Monoclonal antibodies (mAbs) are at present one of the fastest growing products of pharmaceutical industry, with widespread applications in biochemistry, biology, and medicine. The operation of mAbs production processes is predominantly based on empirical knowledge, the improvements being achieved by using trial-and-error experiments and precedent practices. The nonlinearity of these processes and the absence of suitable instrumentation require an enhanced modelling effort and modern kinetic parameter estimation strategies. The present work is dedicated to nonlinear dynamic modelling and parameter estimation for a mammalian cell culture process used for mAb production. By using a dynamical model of such kind of processes, an optimization-based technique for estimation of kinetic parameters in the model of mammalian cell culture process is developed. The estimation is achieved as a result of minimizing an error function by a particle swarm optimization (PSO) algorithm. The proposed estimation approach is analyzed in this work by using a particular model of mammalian cell culture, as a case study, but is generic for this class of bioprocesses. The presented case study shows that the proposed parameter estimation technique provides a more accurate simulation of the experimentally observed process behaviour than reported in previous studies. PMID:25685797

Stabilities and Biological Activities of Vanadium Drugs: What is the Nature of the Active Species?

PubMed

Levina, Aviva; Lay, Peter A

2017-07-18

Diverse biological activities of vanadium(V) drugs mainly arise from their abilities to inhibit phosphatase enzymes and to alter cell signaling. Initial interest focused on anti-diabetic activities but has shifted to anti-cancer and anti-parasitic drugs. V-based anti-diabetics are pro-drugs that release active components (e.g., H 2 VO 4 - ) in biological media. By contrast, V anti-cancer drugs are generally assumed to enter cells intact; however, speciation studies indicate that nearly all drugs are likely to react in cell culture media during in vitro assays and the same would apply in vivo. The biological activities are due to V V and/or V IV reaction products with cell culture media, or the release of ligands (e.g., aromatic diimines, 8-hydroxyquinolines or thiosemicarbazones) that bind to essential metal ions in the media. Careful consideration of the stability and speciation of V complexes in cell culture media and in biological fluids is essential to design targeted V-based anti-cancer therapies. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

NASA Bioculture System: From Experiment Definition to Flight Payload

NASA Technical Reports Server (NTRS)

Sato, Kevin Y.; Almeida, Eduardo; Austin, Edward M.

2014-01-01

Starting in 2015, the NASA Bioculture System will be available to the science community to conduct cell biology and microbiology experiments on ISS. The Bioculture System carries ten environmentally independent Cassettes, which house the experiments. The closed loop fluids flow path subsystem in each Cassette provides a perfusion-based method for maintain specimen cultures in a shear-free environment by using a biochamber based on porous hollow fiber bioreactor technology. Each Cassette contains an incubator and separate insulated refrigerator compartment for storage of media, samples, nutrients and additives. The hardware is capable of fully automated or manual specimen culturing and processing, including in-flight experiment initiation, sampling and fixation, up to BSL-2 specimen culturing, and the ability to up to 10 independent cultures in parallel for statistical analysis. The incubation and culturing of specimens in the Bioculture System is a departure from standard laboratory culturing methods. Therefore, it is critical that the PI has an understanding the pre-flight test required for successfully using the Bioculture System to conduct an on-orbit experiment. Overall, the PI will conduct a series of ground tests to define flight experiment and on-orbit implementation requirements, verify biocompatibility, and determine base bioreactor conditions. The ground test processes for the utilization of the Bioculture System, from experiment selection to flight, will be reviewed. Also, pre-flight test schedules and use of COTS ground test equipment (CellMax and FiberCell systems) and the Bioculture System will be discussed.

Human endothelial cells hollow fiber membrane bioreactor as a model of the blood vessel for in vitro studies.

PubMed

Ciechanowska, Anna; Ladyzynski, Piotr; Hoser, Grazyna; Sabalinska, Stanislawa; Kawiak, Jerzy; Foltynski, Piotr; Wojciechowski, Cezary; Chwojnowski, Andrzej

2016-09-01

Human endothelial cells are used in experimental models for studying in vitro pathophysiological mechanisms of different diseases. We developed an original bioreactor, which can simulate human blood vessel, with capillary polysulfone membranes covered with the human umbilical vein endothelial cells (HUVECs) and we characterized its properties. The elaborated cell seeding and culturing procedures ensured formation of a confluent cell monolayer on the inside surface of capillaries within 24 h of culturing under the shear stress of 6.6 dyn/cm(2). The optimal density of cells to be seeded was 60,000 cells/cm(2). Labeling HUVECs with carboxyfluorescein succinimidyl ester (CFSE) did not influence cells' metabolism. Flow cytometry-based analysis of HUVECs stained with CFSE demonstrated that in a presence of the shear stress cells' proliferation was much inhibited (after 72 h proliferation index was equal to 1.9 and 6.2 for cultures with and without shear stress, respectively) and the monolayer was formed mainly due to migration and spreading of cells that were physiologically elongated in a direction of the flow. Monitoring of cells' metabolism showed that HUVECs cultured in a presence of the shear stress preferred anaerobic metabolism and they consumed 1.5 times more glucose and produced 2.3 times more lactate than the cells cultured under static conditions. Daily von Willebrand factor production by HUVECs was near 2 times higher in a presence of the shear stress. The developed model can be used for at least 3 days in target studies under conditions mimicking the in vivo state more closely than the static HUVEC cultures.

Response of human dental pulp cells to a silver-containing PLGA/TCP-nanofabric as a potential antibacterial regenerative pulp-capping material.

PubMed

Cvikl, Barbara; Hess, Samuel C; Miron, Richard J; Agis, Hermann; Bosshardt, Dieter; Attin, Thomas; Schmidlin, Patrick R; Lussi, Adrian

2017-02-27

Damage or exposure of the dental pulp requires immediate therapeutic intervention. This study assessed the biocompatibility of a silver-containing PLGA/TCP-nanofabric scaffold (PLGA/Ag-TCP) in two in vitro models, i.e. the material adapted on pre-cultured cells and cells directly cultured on the material, respectively. Collagen saffolds with and without hyaluronan acid (Coll-HA; Coll) using both cell culturing methods and cells growing on culture plates served as reference. Cell viability and proliferation were assessed after 24, 48, and 72 h based on formazan formation and BrdU incorporation. Scaffolds were harvested. Gene expression of interleukin(IL)-6, tumor necrosis factor (TNF)-alpha, and alkaline phosphatase (AP) was assessed 24 h after stimulation. In both models formazan formation and BrdU incorporation was reduced by PLGA/Ag-TCP on dental pulp cells, while no significant reduction was found in cells with Coll and Coll-HA. Cells with PLGA/Ag-TCP for 72 h showed similar relative BrdU incorporation than cells stimulated with Coll and Coll-HA. A prominent increase in the pro-inflammatory genes IL-6 and TNF-α was observed when cells were cultured with PLGA/Ag-TCP compared to the other groups. This increase was parallel with a slight increase in AP expression. Overall, no differences between the two culture methods were observed. PLGA/Ag-TCP decreased viability and proliferation rate of human dental pulp cells and increased the pro-inflammatory capacity and alkaline phosphatase expression. Whether these cellular responses observed in vitro translate into pulp regeneration in vivo will be assessed in further studies.

Microspectroscopic investigation of the membrane clogging during the sterile filtration of the growth media for mammalian cell culture.

PubMed

Cao, Xiaolin; Loussaert, James A; Wen, Zai-qing

2016-02-05

Growth media for mammalian cell culture are very complex mixtures of several dozens of ingredients, and thus the preparation of qualified media is critical to viable cell density and final product titers. For liquid media prepared from powdered ingredients, sterile filtration is required prior to use to safeguard the cell culture process. Recently one batch of our prepared media failed to pass through the sterile filtration due to the membrane clogging. In this study, we report the root cause analysis of the failed sterile filtration based on the investigations of both the fouling media and the clogged membranes with multiple microspectroscopic techniques. Cellular particles or fragments were identified in the fouling media and on the surfaces of the clogged membranes, which were presumably introduced to the media from the bacterial contamination. This study demonstrated that microspectroscopic techniques may be used to rapidly identify both microbial particles and inorganic precipitates in the cell culture media. Copyright © 2015 Elsevier B.V. All rights reserved.

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

PubMed

Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

2016-08-01

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

PubMed Central

Muraglia, Anita; Nguyen, Van Thi; Nardini, Marta; Mogni, Massimo; Coviello, Domenico; Dozin, Beatrice; Strada, Paolo; Baldelli, Ilaria; Formica, Matteo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-01-01

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype. PMID:29209609

Multilayer-based lab-on-a-chip systems for perfused cell-based assays

NASA Astrophysics Data System (ADS)

Klotzbach, Udo; Sonntag, Frank; Grünzner, Stefan; Busek, Mathias; Schmieder, Florian; Franke, Volker

2014-12-01

A novel integrated technology chain of laser-microstructured multilayer foils for fast, flexible, and low-cost manufacturing of lab-on-a-chip devices especially for complex cell and tissue culture applications, which provides pulsatile fluid flow within physiological ranges at low media-to-cells ratio, was developed and established. Initially the microfluidic system is constructively divided into individual layers, which are formed by separate foils or plates. Based on the functional boundary conditions and the necessary properties of each layer, their corresponding foils and plates are chosen. In the third step, the foils and plates are laser microstructured and functionalized from both sides. In the fourth and last manufacturing step, the multiple plates and foils are joined using different bonding techniques like adhesive bonding, welding, etc. This multilayer technology together with pneumatically driven micropumps and valves permits the manufacturing of fluidic structures and perfusion systems, which spread out above multiple planes. Based on the established lab-on-a-chip platform for perfused cell-based assays, a multilayer microfluidic system with two parallel connected cell culture chambers was successfully implemented.

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients.

PubMed

Siegler, Uwe; Meyer-Monard, Sandrine; Jörger, Simon; Stern, Martin; Tichelli, André; Gratwohl, Alois; Wodnar-Filipowicz, Aleksandra; Kalberer, Christian P

2010-10-01

Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

NASA Astrophysics Data System (ADS)

Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-08-01

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes.

Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

PubMed Central

Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O’Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-01-01

Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes. PMID:27478033

The selective treatment of clinical mastitis based on on-farm culture results: II. Effects on lactation performance, including clinical mastitis recurrence, somatic cell count, milk production, and cow survival.

PubMed

Lago, A; Godden, S M; Bey, R; Ruegg, P L; Leslie, K

2011-09-01

The objective of this multi-state, multi-herd clinical trial was to report on the efficacy of using an on-farm culture system to guide strategic treatment decisions in cows with clinical mastitis. The study was conducted in 8 commercial dairy farms ranging in size from 144 to 1,795 cows from Minnesota, Wisconsin, and Ontario, Canada. A total of 422 cows affected with mild or moderate clinical mastitis in 449 quarters were randomly assigned to either (1) a positive-control treatment program or (2) an on-farm culture-based treatment program. Quarter cases assigned to the positive-control group received immediate on-label intramammary treatment with cephapirin sodium. Quarters assigned to the culture-based treatment program were not treated until the results of on-farm culture were determined after 18 to 24h of incubation. Quarters in the culture-based treatment program that had gram-positive growth or a mixed infection were treated according to label instruction using intramammary cephapirin sodium. Quarters assigned to the culture-based treatment program that had gram-negative or no-growth did not receive intramammary therapy. It was already reported in a companion paper that the selective treatment of clinical mastitis based on on-farm culture results decreases antibiotic use by half and tends to decrease milk withholding time without affecting short-term clinical and bacteriological outcomes. The present article reports on long-term outcomes of the aforementioned study. No statistically significant differences existed between cases assigned to the positive-control program and cases assigned to the culture-based treatment program in risk and days for recurrence of clinical mastitis in the same quarter (35% and 78 d vs. 43% and 82 d), linear somatic cell count (4.2 vs. 4.4), daily milk production (30.0 vs. 30.7 kg), and risk and days for culling or death events (28% and 160 d vs. 32% and 137 d) for the rest of the lactation after enrollment of the clinical mastitis case. In summary, the selective treatment of clinical mastitis based on on-farm culture resulted in no differences in long-term outcomes, such as recurrence of clinical mastitis in the same quarter, somatic cell count, milk production, and cow survival for the rest of the lactation after clinical mastitis. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

PubMed

Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

2011-11-01

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions. Copyright © 2011 Elsevier Inc. All rights reserved.

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line.

PubMed

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133(+), CD133(-) and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133(+) cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Although CD133(+) derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells.

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line

PubMed Central

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Objective Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student’s t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Conclusion Although CD133+ derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells. PMID:27054115

Cytotoxicity and genotoxicity of natural resin-based experimental endodontic sealers.

PubMed

Silva, Gleyce O; Cavalcanti, Bruno N; Oliveira, Tatiana R; Bin, Claudia V; Camargo, Samira E A; Camargo, Carlos H R

2016-05-01

The development of endodontic sealers based on natural resins seems to be promising, given their improved biological properties. This study evaluated the cytotoxic and genotoxic effects of two experimental root canal sealers, based on extracts from Copaifera multijuga and Ricinus communis (castor oil polymer), comparing them to synthetic resin-based sealers: a single methacrylate-based, a multi-methacrylate-based, and an epoxy resin-based sealers. Sealers were prepared, set, and exposed to cell culture medium for 24 h at 37 °C with CO2. V79 cells were exposed to serial dilutions of the extracts of each sealer for 24 h. Cell viability was measured by the MTT assay and genotoxicity was assessed by the formation of micronuclei. The single methacrylate-based sealer had the most cytotoxic effects, with significant reduction in cell viability in all dilutions of the extract. The castor oil polymer-based sealer was, on the other hand, the most biocompatible sealer, with no cytotoxic effects at any concentration. All tested sealers were not genotoxic, excepting the single methacrylate-based sealer. The tested natural resin-based sealers presented low cytotoxic and no genotoxic effects on cell cultures. These results may suggest a good alternative to develop new endodontic sealers, in order to achieve better biological response and healing, when compared to commercially available sealers.

Microgravity

NASA Image and Video Library

1998-01-01

Cells from kidneys lose some of their special features in conventional culture but form spheres replete with specialized cell microvilli (hair) and synthesize hormones that may be clinically useful. Ground-based research studies have demonstrated that both normal and neoplastic cells and tissues recreate many of the characteristics in the NASA bioreactor that they display in vivo. Proximal kidney tubule cells that normally have rich apically oriented microvilli with intercellular clefts in the kidney do not form any of these structures in conventional two-dimensional monolayer culture. However, when normal proximal renal tubule cells are cultured in three-dimensions in the bioreactor, both the microvilli and the intercellular clefts form. This is important because, when the morphology is recreated, the function is more likely also to be rejuvenated. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC).

Tissue grown in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Cells from kidneys lose some of their special features in conventional culture but form spheres replete with specialized cell microvilli (hair) and synthesize hormones that may be clinically useful. Ground-based research studies have demonstrated that both normal and neoplastic cells and tissues recreate many of the characteristics in the NASA bioreactor that they display in vivo. Proximal kidney tubule cells that normally have rich apically oriented microvilli with intercellular clefts in the kidney do not form any of these structures in conventional two-dimensional monolayer culture. However, when normal proximal renal tubule cells are cultured in three-dimensions in the bioreactor, both the microvilli and the intercellular clefts form. This is important because, when the morphology is recreated, the function is more likely also to be rejuvenated. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC).

Development of scaffold architectures and heterotypic cell systems for hepatocyte transplantation

NASA Astrophysics Data System (ADS)

Alzebdeh, Dalia Abdelrahim

In vitro assembly of functional liver tissue is needed to enable the transplantation of tissue-engineered livers. In addition, there is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of sizable implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability, an issue which is closely related to the high metabolic rate of hepatocytes. In this study, we first compared two designs of highly porous chitosan-heparin scaffolds seeded with hepatocytes in dynamic perfusion bioreactor systems. The aim was to promote cell seeding efficiency by effectively entrapping 100 million hepatocytes at high density. We found that scaffolds with radially tapering pore architecture had highly efficient cell entrapment that maximized donor hepatocyte utilization, compared to alternate pore structures. Hepatocytes showed higher seeding efficiency and metabolic function when seeded as single cell suspensions as opposed to pre-formed, 100microm aggregates. Seeding efficiency was found to increase with flow rate, with single cell and aggregate suspension exhibiting different optimal flow rates. However, metabolic performance results indicated significant shear damage to cells at high efficiency flow rates. To better maintain hepatocyte basement membrane and cell polarity, spheroid co-cultures with mesenchymal stem cells (MSC) were investigated. Hepatocytes and MSCs were seeded in three different architectures in an effort to optimize the spatial arrangement of the two cell types. MSC co-culture greatly enhanced hepatocyte metabolic function in agitated cultures. Interestingly, the effects of diffusion limitations in spheroid culture, coupled with shear damage and subsequent removal of outer hepatocyte layers produced a defined oscillation of urea production rates in certain co-culture arrangements. A mathematical model of urea synthesis in shear-exposed, co-culture spheroids reproduced the metabolic oscillations observed. This result together with culture observations suggests that MSCs can provide both physiological support and some direct shear protection to hepatocytes in perfused or shear-exposed culture environments. Finally, in order to reduce hepatocyte exposure to excessive shear forces in perfused scaffolds, a modular scaffold design based on polyelectrolyte fiber encapsulation was explored. Scaffolds with uniformly distributed, shear protected cells were achieved.

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

Development of Cell Analysis Software for Cultivated Corneal Endothelial Cells.

PubMed

Okumura, Naoki; Ishida, Naoya; Kakutani, Kazuya; Hongo, Akane; Hiwa, Satoru; Hiroyasu, Tomoyuki; Koizumi, Noriko

2017-11-01

To develop analysis software for cultured human corneal endothelial cells (HCECs). Software was designed to recognize cell borders and to provide parameters such as cell density, coefficient of variation, and polygonality of cultured HCECs based on phase contrast images. Cultured HCECs with high or low cell density were incubated with Ca-free and Mg-free phosphate-buffered saline for 10 minutes to reveal the cell borders and were then analyzed with software (n = 50). Phase contrast images showed that cell borders were not distinctly outlined, but these borders became more distinctly outlined after phosphate-buffered saline treatment and were recognized by cell analysis software. The cell density value provided by software was similar to that obtained using manual cell counting by an experienced researcher. Morphometric parameters, such as the coefficient of variation and polygonality, were also produced by software, and these values were significantly correlated with cell density (Pearson correlation coefficients -0.62 and 0.63, respectively). The software described here provides morphometric information from phase contrast images, and it enables subjective and noninvasive quality assessment for tissue engineering therapy of the corneal endothelium.

An approach to collective behavior in cell cultures: modeling and analysis of ECIS data

NASA Astrophysics Data System (ADS)

Rabson, David; Lafalce, Evan; Lovelady, Douglas; Lo, Chun-Min

2011-03-01

We review recent results in which statistical measures of noise in ECIS data distinguished healthy cell cultures from cancerous or poisoned ones: after subtracting the ``signal,'' the 1 /fα noise in the healthy cultures shows longer short-time and long-time correlations. We discuss application of an artificial neural network to detect the cancer signal, and we demonstrate a computational model of cell-cell communication that produces signals similar to those of the experimental data. The simulation is based on the q -state Potts model with inspiration from the Bak-Tang-Wiesenfeld sand-pile model. We view the level of organization larger than cells but smaller than organs or tissues as a kind of ``mesoscopic'' biological physics, in which few-body interactions dominate, and the experiments and computational model as ways of exploring this regime.

Characterization of Rat Meibomian Gland Ion and Fluid Transport

PubMed Central

Yu, Dongfang; Davis, Richard M.; Aita, Megumi; Burns, Kimberlie A.; Clapp, Phillip W.; Gilmore, Rodney C.; Chua, Michael; O'Neal, Wanda K.; Schlegel, Richard; Randell, Scott H.; C. Boucher, Richard

2016-01-01

Purpose We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. Methods Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of βENaC, the Na+/K+/Cl− cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays. Results Expression of multiple ion channel/transporter genes was detected in rat MG tissues. β-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels. Conclusions This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established. PMID:27127933

Analysis of type II diabetes mellitus adipose-derived stem cells for tissue engineering applications

PubMed Central

Minteer, Danielle Marie; Young, Matthew T; Lin, Yen-Chih; Over, Patrick J; Rubin, J Peter; Gerlach, Jorg C

2015-01-01

To address the functionality of diabetic adipose-derived stem cells in tissue engineering applications, adipose-derived stem cells isolated from patients with and without type II diabetes mellitus were cultured in bioreactor culture systems. The adipose-derived stem cells were differentiated into adipocytes and maintained as functional adipocytes. The bioreactor system utilizes a hollow fiber–based technology for three-dimensional perfusion of tissues in vitro, creating a model in which long-term culture of adipocytes is feasible, and providing a potential tool useful for drug discovery. Daily metabolic activity of the adipose-derived stem cells was analyzed within the medium recirculating throughout the bioreactor system. At experiment termination, tissues were extracted from bioreactors for immunohistological analyses in addition to gene and protein expression. Type II diabetic adipose-derived stem cells did not exhibit significantly different glucose consumption compared to adipose-derived stem cells from patients without type II diabetes (p > 0.05, N = 3). Expression of mature adipocyte genes was not significantly different between diabetic/non-diabetic groups (p > 0.05, N = 3). Protein expression of adipose tissue grown within all bioreactors was verified by Western blotting.The results from this small-scale study reveal adipose-derived stem cells from patients with type II diabetes when removed from diabetic environments behave metabolically similar to the same cells of non-diabetic patients when cultured in a three-dimensional perfusion bioreactor, suggesting that glucose transport across the adipocyte cell membrane, the hindrance of which being characteristic of type II diabetes, is dependent on environment. The presented observation describes a tissue-engineered tool for long-term cell culture and, following future adjustments to the culture environment and increased sample sizes, potentially for anti-diabetic drug testing. PMID:26090087

Development of a dose-controlled multiculture cell exposure chamber for efficient delivery of airborne and engineered nanoparticles

NASA Astrophysics Data System (ADS)

Asimakopoulou, Akrivi; Daskalos, Emmanouil; Lewinski, Nastassja; Riediker, Michael; Papaioannou, Eleni; Konstandopoulos, Athanasios G.

2013-04-01

In order to study the various health influencing parameters related to engineered nanoparticles as well as to soot emitted by Diesel engines, there is an urgent need for appropriate sampling devices and methods for cell exposure studies that simulate the respiratory system and facilitate associated biological and toxicological tests. The objective of the present work was the further advancement of a Multiculture Exposure Chamber (MEC) into a dose-controlled system for efficient delivery of nanoparticles to cells. It was validated with various types of nanoparticles (Diesel engine soot aggregates, engineered nanoparticles for various applications) and with state-of-the-art nanoparticle measurement instrumentation to assess the local deposition of nanoparticles on the cell cultures. The dose of nanoparticles to which cell cultures are being exposed was evaluated in the normal operation of the in vitro cell culture exposure chamber based on measurements of the size specific nanoparticle collection efficiency of a cell free device. The average efficiency in delivering nanoparticles in the MEC was approximately 82%. The nanoparticle deposition was demonstrated by Transmission Electron Microscopy (TEM). Analysis and design of the MEC employs Computational Fluid Dynamics (CFD) and true to geometry representations of nanoparticles with the aim to assess the uniformity of nanoparticle deposition among the culture wells. Final testing of the dose-controlled cell exposure system was performed by exposing A549 lung cell cultures to fluorescently labeled nanoparticles. Delivery of aerosolized nanoparticles was demonstrated by visualization of the nanoparticle fluorescence in the cell cultures following exposure. Also monitored was the potential of the aerosolized nanoparticles to generate reactive oxygen species (ROS) (e.g. free radicals and peroxides generation), thus expressing the oxidative stress of the cells which can cause extensive cellular damage or damage on DNA.

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro.

PubMed

Kim, Suna; Piao, Jiyuan; Son, Youngsook; Hong, Hyun Sook

2017-03-25

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culture are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. Copyright © 2017 Elsevier Inc. All rights reserved.

PCR amplification and genetic analysis in a microwell cell culturing chip.

PubMed

Lindström, Sara; Hammond, Maria; Brismar, Hjalmar; Andersson-Svahn, Helene; Ahmadian, Afshin

2009-12-21

We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity.

Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation.

PubMed

Akbarian, Vahe; Wang, Weijia; Audet, Julie

2012-05-01

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture. Copyright © 2012 International Society for Advancement of Cytometry.

Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state.

PubMed

Zhang, Kehua; Cai, Zhe; Li, Yang; Shu, Jun; Pan, Lin; Wan, Fang; Li, Hong; Huang, Xiaojie; He, Chun; Liu, Yanqiu; Cui, Xiaohui; Xu, Yang; Gao, Yan; Wu, Liqun; Cao, Shanxia; Li, Lingsong

2011-08-01

Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

Characterization of human pancreatic progenitor cells.

PubMed

Noguchi, Hirofumi; Naziruddin, Bashoo; Jackson, Andrew; Shimoda, Masayuki; Ikemoto, Tetsuya; Fujita, Yasutaka; Chujo, Daisuke; Takita, Morihito; Kobayashi, Naoya; Onaca, Nicholas; Hayashi, Shuji; Levy, Marlon F; Matsumoto, Shinichi

2010-01-01

β-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for β-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed "cobblestone" morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed "fibroblast-like" morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6-10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.

Model-based strategy for cell culture seed train layout verified at lab scale.

PubMed

Kern, Simon; Platas-Barradas, Oscar; Pörtner, Ralf; Frahm, Björn

2016-08-01

Cell culture seed trains-the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale-are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system.

PubMed

Kim, Nan-Sun; Yu, Hwa-Young; Chung, Nguyen-Duc; Kwon, Tae-Ho; Yang, Moon-Sik

2014-09-01

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8-4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid. The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53-39 mg/L per cycle during five recycling cycles. Copyright © 2014 Elsevier Inc. All rights reserved.

Distribution of culturable microorganisms in Fennoscandian Shield groundwater.

PubMed

Haveman, Shelley A; Pedersen, Karsten

2002-02-01

Microbial populations in 16 groundwater samples from six Fennoscandian Shield sites in Finland and Sweden were investigated. The average total cell number was 3.7x10(5) cells ml(-1), and there was no change in the mean of the total cell numbers to a depth of 1390 m. Culture media were designed based on the chemical composition of each groundwater sample and used successfully to culture anaerobic microorganisms from all samples between 65 and 1350 m depth. Between 0.0084 and 14.8% of total cells were cultured from groundwater samples. Sulfate-reducing bacteria, iron-reducing bacteria and heterotrophic acetogenic bacteria were cultured from groundwater sampled at 65-686 m depth in geographically distant sites. Different microbial populations were cultured from deeper, older and more saline groundwater from 863 to 1350 m depth. Principal component analysis of groundwater chemistry data showed that sulfate- and iron-reducing bacteria were not detected in the most saline groundwater. Iron-reducing bacteria and acetogens were cultured from deep groundwater that contained 0.35-3.5 mM sulfate, while methanogens and acetogens were cultured from deep sulfate-depleted groundwater. In one borehole from which autotrophic methanogens were cultured, dissolved inorganic carbon was enriched in (13)C compared to other Fennoscandian Shield groundwater samples, suggesting that autotrophs were active. It can be concluded that a diverse microbial community is present from the surface to over 1300 m depth in the Fennoscandian Shield.