Making microenvironments: A look into incorporating macromolecular crowding into in vitro experiments, to generate biomimetic microenvironments which are capable of directing cell function for tissue engineering applications.

PubMed

Benny, Paula; Raghunath, Michael

2017-01-01

Biomimetic microenvironments are key components to successful cell culture and tissue engineering in vitro. One of the most accurate biomimetic microenvironments is that made by the cells themselves. Cell-made microenvironments are most similar to the in vivo state as they are cell-specific and produced by the actual cells which reside in that specific microenvironment. However, cell-made microenvironments have been challenging to re-create in vitro due to the lack of extracellular matrix composition, volume and complexity which are required. By applying macromolecular crowding to current cell culture protocols, cell-made microenvironments, or cell-derived matrices, can be generated at significant rates in vitro. In this review, we will examine the causes and effects of macromolecular crowding and how it has been applied in several in vitro systems including tissue engineering.

Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

PubMed

Baudoin, Régis; Corlu, Anne; Griscom, Laurent; Legallais, Cécile; Leclerc, Eric

2007-06-01

Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver/lung organ model.

Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application.

PubMed

Takayama, Naoya; Eto, Koji

2012-10-01

Human pluripotent stem cells [PSCs; including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] can infinitely proliferate in vitro and are easily accessible for gene manipulation. Megakaryocytes (MKs) and platelets can be created from human ESCs and iPSCs in vitro and represent a potential source of blood cells for transfusion and a promising tool for studying the human thrombopoiesis. Moreover, disease-specific iPSCs are a powerful tool for elucidating the pathogenesis of hematological diseases and for drug screening. In that context, we and other groups have developed in vitro MK and platelet differentiation systems from human pluripotent stem cells (PSCs). Combining this co-culture system with a drug-inducible gene expression system enabled us to clarify the novel role played by c-MYC during human thrombopoiesis. In the next decade, technical advances (e.g., high-throughput genomic sequencing) will likely enable the identification of numerous gene mutations associated with abnormal thrombopoiesis. Combined with such technology, an in vitro system for differentiating human PSCs into MKs and platelets could provide a novel platform for studying human gene function associated with thrombopoiesis.

Biomarkers of cell senescence

DOEpatents

Dimri, G.P.; Campisi, J.; Peacocke, M.

1998-08-18

The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, {beta}-galactosidase activity is utilized as a means by which cell senescence may be assessed either in vitro cell cultures or in vivo. 1 fig.

Biomarkers of cell senescence

DOEpatents

Dirmi, G.P.; Campisi, J.; Peacocke, M.

1996-02-13

The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, {beta}-galactosidase activity is utilized as a means by which cell senescence may be assessed either in in vitro cell cultures or in vivo. 1 fig.

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

PubMed

Ohkoshi, Katsuhiro; Takahashi, Seiya; Koyama, Shin-Ichiro; Akagi, Satoshi; Adachi, Noritaka; Furusawa, Tadashi; Fujimoto, Jun-Ichiro; Takeda, Kumiko; Kubo, Masanori; Izaike, Yoshiaki; Tokunaga, Tomoyuki

2003-01-01

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

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.

Comparative Iron Oxide Nanoparticle Cellular Dosimetry and Response in Mice by the Inhalation and Liquid Cell Culture Exposure Routes

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

Teeguarden, Justin G.; Mikheev, Vladimir B.; Minard, Kevin R.

testing the rapidly growing number of nanomaterials requires large scale use of in vitro systems under the presumption that these systems are sufficiently predictive or descriptive of responses in in vivo systems for effective use in hazard ranking. We hypothesized that improved relationships between in vitro and in vivo models of experimental toxicology for nanomaterials would result from placing response data in vitro and in vivo on the same dose scale, the amount of material associated with cells (target cell dose). Methods: Balb/c mice were exposed nose-only to an aerosol of 12.8 nm (68.6 nm CMD, 19.9 mg/m3, 4 hours)more » super paramagnetic iron oxide particles, target cell doses were calculated and biomarkers of response anchored with histological evidence were identified by global transcriptomics. Representative murine epithelial and macrophage cell types were exposed in vitro to the same material in liquid suspension for four hours and levels nanoparticle regulated cytokine transcripts identified in vivo were quantified as a function of measured nanoparticle cellular dose. Results. Target tissue doses of 0.009-0.4 μg SPIO/cm2 lung led to an inflammatory response in the alveolar region characterized by interstitial inflammation and macrophage infiltration. In vitro, higher target tissue doses of ~1.2-4 μg SPIO/ cm2 of cells were required to induce transcriptional regulation of markers of inflammation, CXCL2 CCL3, in C10 lung epithelial cells. Estimated in vivo macrophage SPIO nanoparticle doses ranged from 1-100 pg/cell, and induction of inflammatory markers was observed in vitro in macrophages at doses of 8-35 pg/cell. Conclusions: Application of target tissue dosimetry revealed good correspondence between target cell doses triggering inflammatory processes in vitro and in vivo in the alveolar macrophage population, but not in the epithelial cells of the alveolar region. These findings demonstrate the potential for target tissue dosimetry to enable the more quantitative comparison of in vitro and in vivo systems advance their use for hazard assessment and extrapolation to humans. The mildly inflammogentic cellular doses experienced by mice were similar those calculated for humans exposed to the same at the existing permissible exposure limit of 10 mg/m3 iron oxide (as Fe).« less

Evaluation of intratympanic formulations for inner ear delivery: methodology and sustained release formulation testing

PubMed Central

Liu, Hongzhuo; Feng, Liang; Tolia, Gaurav; Liddell, Mark R.; Hao, Jinsong; Li, S. Kevin

2013-01-01

A convenient and efficient in vitro diffusion cell method to evaluate formulations for inner ear delivery via the intratympanic route is currently not available. The existing in vitro diffusion cell systems commonly used to evaluate drug formulations do not resemble the physical dimensions of the middle ear and round window membrane. The objectives of this study were to examine a modified in vitro diffusion cell system of a small diffusion area for studying sustained release formulations in inner ear drug delivery and to identify a formulation for sustained drug delivery to the inner ear. Four formulations and a control were examined in this study using cidofovir as the model drug. Drug release from the formulations in the modified diffusion cell system was slower than that in the conventional diffusion cell system due to the decrease in the diffusion surface area of the modified diffusion cell system. The modified diffusion cell system was able to show different drug release behaviors among the formulations and allowed formulation evaluation better than the conventional diffusion cell system. Among the formulations investigated, poly(lactic-co-glycolic acid)–poly(ethylene glycol)–poly(lactic-co-glycolic acid) triblock copolymer systems provided the longest sustained drug delivery, probably due to their rigid gel structures and/or polymer-to-cidofovir interactions. PMID:23631539

Micropatterning of cells reveals chiral morphogenesis

PubMed Central

2013-01-01

Invariant left-right (LR) patterning or chirality is critical for embryonic development. The loss or reversal of LR asymmetry is often associated with malformations and disease. Although several theories have been proposed, the exact mechanism of the initiation of the LR symmetry has not yet been fully elucidated. Recently, chirality has been detected within single cells as well as multicellular structures using several in vitro approaches. These studies demonstrated the universality of cell chirality, its dependence on cell phenotype, and the role of physical boundaries. In this review, we discuss the theories for developmental LR asymmetry, compare various in vitro cell chirality model systems, and highlight possible roles of cell chirality in stem cell differentiation. We emphasize that the in vitro cell chirality systems have great promise for helping unveil the nature of chiral morphogenesis in development. PMID:23672821

In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses.

PubMed

Chen, Ying; Zhou, Wenda; Roh, Terrence; Estes, Mary K; Kaplan, David L

2017-01-01

There is a need for functional in vitro 3D human intestine systems that can bridge the gap between conventional cell culture studies and human trials. The successful engineering in vitro of human intestinal tissues relies on the use of the appropriate cell sources, biomimetic scaffolds, and 3D culture conditions to support vital organ functions. We previously established a compartmentalized scaffold consisting of a hollow space within a porous bulk matrix, in which a functional and physiologically relevant intestinal epithelium system was generated using intestinal cell lines. In this study, we adopt the 3D scaffold system for the cultivation of stem cell-derived human small intestinal enteriods (HIEs) to engineer an in vitro 3D model of a nonstransformed human small intestinal epithelium. Characterization of tissue properties revealed a mature HIE-derived epithelium displaying four major terminally differentiated epithelial cell types (enterocytes, Goblet cells, Paneth cells, enteroendocrine cells), with tight junction formation, microvilli polarization, digestive enzyme secretion, and low oxygen tension in the lumen. Moreover, the tissue model demonstrates significant antibacterial responses to E. coli infection, as evidenced by the significant upregulation of genes involved in the innate immune response. Importantly, many of these genes are activated in human patients with inflammatory bowel disease (IBD), implicating the potential application of the 3D stem-cell derived epithelium for the in vitro study of host-microbe-pathogen interplay and IBD pathogenesis.

Application of cell co-culture system to study fat and muscle cells.

PubMed

Pandurangan, Muthuraman; Hwang, Inho

2014-09-01

Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

Setting the proportion of CD4+ and CD8+ T-cells co-cultured with canine macrophages infected with Leishmania chagasi.

PubMed

Viana, Kelvinson Fernandes; Aguiar-Soares, Rodrigo Dian Oliveira; Ker, Henrique Gama; Resende, Lucilene Aparecida; Souza-Fagundes, Elaine Maria; Dutra, Walderez Ornelas; Fujiwara, Ricardo Toshio; da Silveira-Lemos, Denise; Sant'Ana, Rita de Cássia Oliveira; Wardini, Amanda Brito; Araújo, Márcio Sobreira Silva; Martins-Filho, Olindo Assis; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

2015-07-30

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions. Copyright © 2015 Elsevier B.V. All rights reserved.

Deciphering defective amelogenesis using in vitro culture systems.

PubMed

Arinawati, Dian Yosi; Miyoshi, Keiko; Tanimura, Ayako; Horiguchi, Taigo; Hagita, Hiroko; Noma, Takafumi

2018-04-01

The conventional two-dimensional (2D) in vitro culture system is frequently used to analyze the gene expression with or without extracellular signals. However, the cells derived from primary culture and cell lines frequently deviate the gene expression profile compared to the corresponding in vivo samples, which sometimes misleads the actual gene regulation in vivo. To overcome this gap, we developed the comparative 2D and 3D in vitro culture systems and applied them to the genetic study of amelogenesis imperfecta (AI) as a model. Recently, we found specificity protein 6 (Sp6) mutation in an autosomal-recessive AI rat that was previously named AMI. We constructed 3D structure of ARE-B30 cells (AMI-derived rat dental epithelial cells) or G5 (control wild type cells) combined with RPC-C2A cells (rat pulp cell line) separated by the collagen membrane, while in 2D structure, ARE-B30 or G5 was cultured with or without the collagen membrane. Comparative analysis of amelogenesis-related gene expression in ARE-B30 and G5 using our 2D and 3D in vitro systems revealed distinct expression profiles, showing the causative outcomes. Bone morphogenetic protein 2 and follistatin were reciprocally expressed in G5, but not in ARE-B30 cells. All-or-none expression of amelotin, kallikrein-related peptidase 4, and nerve growth factor receptor was observed in both cell types. In conclusion, our in vitro culture systems detected the phenotypical differences in the expression of the stage-specific amelogenesis-related genes. Parallel analysis with 2D and 3D culture systems may provide a platform to understand the molecular basis for defective amelogenesis caused by Sp6 mutation. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A quantitative framework to evaluate modeling of cortical development by neural stem cells

PubMed Central

Stein, Jason L.; de la Torre-Ubieta, Luis; Tian, Yuan; Parikshak, Neelroop N.; Hernandez, Israel A.; Marchetto, Maria C.; Baker, Dylan K.; Lu, Daning; Hinman, Cassidy R.; Lowe, Jennifer K.; Wexler, Eric M.; Muotri, Alysson R.; Gage, Fred H.; Kosik, Kenneth S.; Geschwind, Daniel H.

2014-01-01

Summary Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease. PMID:24991955

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro

PubMed Central

Manczinger, Máté; Bocsik, Alexandra; Kocsis, Gabriella F.; Vörös, Andrea; Hegedűs, Zoltán; Marton, Annamária; Vízler, Csaba; Tubak, Vilmos; Deli, Mária; Kemény, Lajos; Nagy, István; Lakatos, Lóránt

2015-01-01

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis. PMID:26366412

Role of Microenvironment in Glioma Invasion: What We Learned from In Vitro Models

PubMed Central

Manini, Ivana; Caponnetto, Federica; Bartolini, Anna; Ius, Tamara; Mariuzzi, Laura; Di Loreto, Carla; Cesselli, Daniela

2018-01-01

The invasion properties of glioblastoma hamper a radical surgery and are responsible for its recurrence. Understanding the invasion mechanisms is thus critical to devise new therapeutic strategies. Therefore, the creation of in vitro models that enable these mechanisms to be studied represents a crucial step. Since in vitro models represent an over-simplification of the in vivo system, in these years it has been attempted to increase the level of complexity of in vitro assays to create models that could better mimic the behaviour of the cells in vivo. These levels of complexity involved: 1. The dimension of the system, moving from two-dimensional to three-dimensional models; 2. The use of microfluidic systems; 3. The use of mixed cultures of tumour cells and cells of the tumour micro-environment in order to mimic the complex cross-talk between tumour cells and their micro-environment; 4. And the source of cells used in an attempt to move from commercial lines to patient-based models. In this review, we will summarize the evidence obtained exploring these different levels of complexity and highlighting advantages and limitations of each system used. PMID:29300332

COMPARISON OF MEDIUM CONCENTRATION VS. ACTUAL TISSUE DOSE IN IN VITRO NEUROTOXICANT MODELS.

EPA Science Inventory

In vitro methods have long been used to model the effects of toxicants on the nervous system. Generally, it is assumed that concentrations of toxicant present in the medium surrounding cells in in vitro models are an adequate biomarker of cell or tissue levels. However, this assu...

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

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

Rasey, J.S.; Nelson, N.J.

1983-01-01

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose.more » The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.« less

Generation of a Close-to-Native In Vitro System to Study Lung Cells-Extracellular Matrix Crosstalk.

PubMed

Garlíková, Zuzana; Silva, Ana Catarina; Rabata, Anas; Potěšil, David; Ihnatová, Ivana; Dumková, Jana; Koledová, Zuzana; Zdráhal, Zbyněk; Vinarský, Vladimír; Hampl, Aleš; Pinto-do-Ó, Perpétua; Nascimento, Diana Santos

2018-01-01

Extracellular matrix (ECM) is an essential component of the tissue microenvironment, actively shaping cellular behavior. In vitro culture systems are often poor in ECM constituents, thus not allowing for naturally occurring cell-ECM interactions. This study reports on a straightforward and efficient method for the generation of ECM scaffolds from lung tissue and its subsequent in vitro application using primary lung cells. Mouse lung tissue was subjected to decellularization with 0.2% sodium dodecyl sulfate, hypotonic solutions, and DNase. Resultant ECM scaffolds were devoid of cells and DNA, whereas lung ECM architecture of alveolar region and blood and airway networks were preserved. Scaffolds were predominantly composed of core ECM and ECM-associated proteins such as collagens I-IV, nephronectin, heparan sulfate proteoglycan core protein, and lysyl oxidase homolog 1, among others. When homogenized and applied as coating substrate, ECM supported the attachment of lung fibroblasts (LFs) in a dose-dependent manner. After ECM characterization and biocompatibility tests, a novel in vitro platform for three-dimensional (3D) matrix repopulation that permits live imaging of cell-ECM interactions was established. Using this system, LFs colonized the ECM scaffolds, displaying a close-to-native morphology in intimate interaction with the ECM fibers, and showed nuclear translocation of the mechanosensor yes-associated protein (YAP), when compared with cells cultured in two dimensions. In conclusion, we developed a 3D-like culture system, by combining an efficient decellularization method with a live-imaging culture platform, to replicate in vitro native lung cell-ECM crosstalk. This is a valuable system that can be easily applied to other organs for ECM-related drug screening, disease modeling, and basic mechanistic studies.

Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases.

PubMed

Ahn, Misol; Kalume, Franck; Pitstick, Rose; Oehler, Abby; Carlson, George; DeArmond, Stephen J

2016-03-01

Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases. Second, drug efficacy may differ between dividing and stationary cells, the latter resembling nondividing neurons in the CNS. Brain aggregates (BrnAggs) derived from embryonic day 15 gestation mouse embryos may represent neuropathogenic processes in prion disease and reflect in vivo drug efficacy. Here, we report a new method for the production of BrnAggs suitable for drug screening and suggest that BrnAggs can model additional neurological diseases such as tauopathies. We also report a functional assay with BrnAggs by measuring electrophysiological activities. Our data suggest that BrnAggs could serve as an effective in vitro cell culture system for drug discovery for neurodegenerative diseases. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

PubMed

Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

2010-01-01

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.

PubMed Central

Sakalian, M; Parker, S D; Weldon, R A; Hunter, E

1996-01-01

The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly. PMID:8648705

Genetic modification of cells for transplantation.

PubMed

Lai, Yi; Drobinskaya, Irina; Kolossov, Eugen; Chen, Chunguang; Linn, Thomas

2008-01-14

Progress in gene therapy has produced promising results that translate experimental research into clinical treatment. Gene modification has been extensively employed in cell transplantation. The main barrier is an effective gene delivery system. Several viral vectors were utilized in end-stage differentiated cells. Recently, successful applications were described with adenovirus-associated vectors. As an alternative, embryonic stem cell- and stem cell-like systems were established for generation of tissue-specified gene-modified cells. Owing to the feasibility for genetic manipulations and the self-renewing potency of these cells they can be used in a way enabling large-scale in vitro production. This approach offers the establishment of in vitro cell culture systems that will deliver sufficient amounts of highly purified, immunoautologous cells suitable for application in regenerative medicine. In this review, the current technology of gene delivery systems to cells is recapitulated and the latest developments for cell transplantation are discussed.

Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants

PubMed Central

Vagasi, Alexandra S.; Rahman, Mohammad M.; Chaudhari, Snehal N.; Kipreos, Edward T.

2017-01-01

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016. PMID:28868332

In vitro generation of viral-antigen dependent cytotoxic T-cells from ginbuna crucian carp, Carassius auratus langsdorfii

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

Somamoto, Tomonori, E-mail: somamoto@agr.kyushu-u.ac.j; Okamoto, Nobuaki; Nakanishi, Teruyuki

2009-06-20

Little is known about antigen-specific T-cell responses to viruses in teleosts due to a lack of a suitable experimental system using inbred or clonal animals. In the present study we have successfully induced an in vitro generation of virus-specific cytotoxic T-cells (CTLs) from isogeneic ginbuna crucian carp. Responder cells (primarily lymphocytes) from crucian carp haematopoietic necrosis virus (CHNV)-infected fish were capable of proliferating after stimulation in vitro with CHNV-infected syngeneic stimulator cells (primarily lymphocytes and macrophages). The effector cells collected 8 and 12 days after the in vitro stimulation efficiently lysed CHNV-infected syngeneic cells, but not CHNV-infected allogeneic cells ormore » different virus (EVA)-infected syngeneic cells. Furthermore, in situ hybridization analysis showed that some effector cells binding to a CHNV-infected target were TCRbeta or CD8alpha positive. These results provide evidence that the teleost effector cells generated in vitro correspond to virus-specific CTL and they recognize virus-infected target cells in a similar manner of mammalian counterparts.« less

A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes.

PubMed

Mueller, A J; Tew, S R; Vasieva, O; Clegg, P D; Canty-Laird, E G

2016-09-27

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

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

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

Stem cells, in vitro gametogenesis and male fertility.

PubMed

Nagamatsu, Go; Hayashi, Katsuhiko

2017-12-01

Reconstitution in culture of biological processes, such as differentiation and organization, is a key challenge in regenerative medicine, and one in which stem cell technology plays a central role. Pluripotent stem cells and spermatogonial stem cells are useful materials for reconstitution of germ cell development in vitro , as they are capable of differentiating into gametes. Reconstitution of germ cell development, termed in vitro gametogenesis, will provide an experimental platform for a better understanding of germ cell development, as well as an alternative source of gametes for reproduction, with the potential to cure infertility. Since germ cells are the cells for 'the next generation', both the culture system and its products must be carefully evaluated. In this issue, we summarize the progress in in vitro gametogenesis, most of which has been made using mouse models, as well as the future challenges in this field. © 2017 Society for Reproduction and Fertility.

Targeted antitumoral dehydrocrotonin nanoparticles with L-ascorbic acid 6-stearate.

PubMed

Frungillo, Lucas; Martins, Dorival; Teixeira, Sérgio; Anazetti, Maristela Conti; Melo, Patrícia da Silva; Durán, Nelson

2009-12-01

Tumoral cells are known to have a higher ascorbic acid uptake than normal cells. Therefore, the aim of this study was to obtain polymeric nanoparticles containing the antitumoral compound trans-dehydrocrotonin (DHC) functionalized with L-ascorbic acid 6-stearate (AAS) to specifically target this system tumoral cells. Nanoparticle suspensions (NP-AAS-DHC) were prepared by the nanoprecipitation method. The systems were characterized for AAS presence by thin-layer chromatography and for drug loading (81-88%) by UV-Vis spectroscopy. To further characterize these systems, in vitro release kinetics, size distribution (100-140 nm) and Zeta potential by photon-correlation spectroscopic method were used. In vitro toxicity against HL60 cells was evaluated by tetrazolium reduction and Trypan blue exclusion assays. Cell death by apoptosis was quantified and characterized by flow cytometry and caspase activity. Zeta potential analyses showed that the system has a negatively charged outer surface and also indicate that AAS is incorporated on the external surface of the nanoparticles. In vitro release kinetics assay showed that DHC loaded in nanoparticles had sustained release behavior. In vitro toxicity assays showed that NP-AAS-DHC suspension was more effective as an antitumoral than free DHC or NP-DHC and increased apoptosis induction by receptor-mediated pathway. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

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

Longitudinal growth of skeletal myotubes in vitro in a new horizontal mechanical cell stimulator

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Karlisch, Patricia

1989-01-01

A tissue-culture model system for growing skeletal-muscle cells under more dynamic conditions than found in normal tissue-culture environments is described. A computerized device presented allows mechanical stimulation of the cell's substratum by 300 to 400 pct in length in the horizontal plane. Cell growth rates and skeletal-muscle organogenesis are stimulated in this in vitro system. It is noted that longitudinal myotube growth observed is accompanied by increased rates of cell proliferation and myoblast fusion. Prestretching the collagen-coated substratum before cell plating is shown to lead to increased cell proliferation, myotube orientation, and longitudinal myotube growth. The effects of substratum stretching on myogenesis in the model system are also assessed and attributed to alterations in the cell's extracellular matrix.

Wnt1a maintains characteristics of dermal papilla cells that induce mouse hair regeneration in a 3D preculture system.

PubMed

Dong, Liang; Hao, Haojie; Liu, Jiejie; Tong, Chuan; Ti, Dongdong; Chen, Deyun; Chen, Li; Li, Meirong; Liu, Huiling; Fu, Xiaobing; Han, Weidong

2017-05-01

Hair follicle morphogenesis and regeneration depend on intensive but well-orchestrated interactions between epithelial and mesenchymal components. Therefore, an alternative strategy to reproduce the process of epithelial-mesenchymal interaction in vitro could use a 3D system containing appropriate cell populations. The 3D air-liquid culture system for reproducibly generating hair follicles from dissociated epithelial and dermal papilla (DP) cells combined with a collagen-chitosan scaffold is described in this study. Wnt-CM was prepared from the supernatant of Wnt1a-expressing bone marrow mesenchymal stem cells (BM-MSCs) that maintain the hair-inducing gene expression of DP cells. The collagen-chitosan scaffold cells (CCS cells) were constructed using a two-step method by inoculating the Wnt-CM-treated DP cells and epidermal (EP) cells into the CCS. The cells in the air-liquid culture formed dermal condensates and a proliferative cell layer in vitro. The CCS cells were able to induce hair regeneration in nude mice. The results demonstrate that Wnt-CM can maintain the hair induction ability of DP cells in expansion cultures, and this approach can be used for large-scale preparation of CCS cells in vitro to treat hair loss. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

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

Lewis, Daniel D.; Department of Biomedical Engineering, University of California Davis, Davis, CA; Villarreal, Fernando D.

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with amore » special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.« less

Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

PubMed Central

Lewis, Daniel D.; Villarreal, Fernando D.; Wu, Fan; Tan, Cheemeng

2014-01-01

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems. PMID:25538941

Synthetic biology outside the cell: linking computational tools to cell-free systems.

PubMed

Lewis, Daniel D; Villarreal, Fernando D; Wu, Fan; Tan, Cheemeng

2014-01-01

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.

A Heterogeneous In Vitro Three Dimensional Model of Tumour-Stroma Interactions Regulating Sprouting Angiogenesis

PubMed Central

Correa de Sampaio, Pedro; Auslaender, David; Krubasik, Davia; Failla, Antonio Virgilio; Skepper, Jeremy N.; Murphy, Gillian; English, William R.

2012-01-01

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model – a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis. PMID:22363483

Intra-hydrogel culture prevents transformation of mesenchymal stem cells induced by monolayer expansion.

PubMed

Jiang, Tongmeng; Liu, Junting; Ouyang, Yiqiang; Wu, Huayu; Zheng, Li; Zhao, Jinmin; Zhang, Xingdong

2018-05-01

In this study, we report that the intra-hydrogel culture system mitigates the transformation of mesenchymal stem cells (MSCs) induced by two-dimensional (2D) expansion. MSCs expanded in monolayer culture prior to encapsulation in collagen hydrogels (group eMSCs-CH) featured impaired stemness in chondrogenesis, comparing with the freshly isolated bone marrow mononuclear cells seeded directly in collagen hydrogels (group fMSCs-CH). The molecular mechanism of the in vitro expansion-triggered damage to MSCs was detected through genome-wide microarray analysis. Results indicated that pathways such as proteoglycans in cancer and pathways in cancer expansion were highly enriched in eMSCs-CH. And multiple up-regulated oncoma-associated genes were verified in eMSCs-CH compared with fMSCs-CH, indicating that expansion in vitro triggered cellular transformation was associated with signaling pathways related to tumorigenicity. Besides, focal adhesion (FA) and mitogen-activated protein kinase (MAPK) signaling pathways were also involved in in vitro expansion, indicating restructuring of the cell architecture. Thus, monolayer expansion in vitro may contribute to vulnerability of MSCs through the regulation of FA and MAPK. This study indicates that intra-hydrogel culture can mitigate the monolayer expansion induced transformation of MSCs and maintain the uniformity of the stem cells, which is a viable in vitro culture system for stem cell therapy.

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

Induction and repair of DNA double-strand breaks in hippocampal neurons of mice of different age after exposure to 60Co γ-rays in vivo and in vitro

NASA Astrophysics Data System (ADS)

Kozhina, R. A.; Chausov, V. N.; Kuzmina, E. A.; Boreyko, A. V.

2018-04-01

One of the central problems of modern radiobiology is the study of DNA damage induction and repair mechanisms in central nervous system cells, in particular, in hippocampal cells. The study of the regularities of molecular damage formation and repair in the hippocampus cells is of special interest, because these cells, unlike most cells of the central nervous system (CNS), keep proliferative activity, i.e. ability to neurogenesis. Age-related changes in hippocampus play an important role, which could lead to radiosensitivity changes in neurons to the ionizing radiation exposure. Regularities in DNA double-strand breaks (DSB) induction and repair in different aged mice hippocampal cells in vivo and in vitro under the action of γ-rays 60Co were studied with DNA comet-assay. The obtained dose dependences of DNA DSB induction are linear both in vivo and in vitro. It is established that in young animals' cells, the degree of DNA damage is higher than in older animals. It is shown that repair kinetics is basically different for exposure in vivo and in vitro.

Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

PubMed

Zeigerer, Anja; Wuttke, Anne; Marsico, Giovanni; Seifert, Sarah; Kalaidzidis, Yannis; Zerial, Marino

2017-01-01

Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Assessment of the Relative Toxicity of N,N-Dipropylcyclohexanecarboxamide, AI3-36326.

DTIC Science & Technology

1983-04-01

cells with or without an in vitro metabolic activation system. The in vitro metabolic activation system was composed of rat liver enzymes and an energy...producing system. The enzymes were contained in a preparation of liver microsomes (S9 fraction)JI fron rats treated with an alkylating agent, Aroclor...to induce enzymes capable of transforming chemicals to more active forms. Cells were examined 10 to 12 hours following treatment when entering mitosis

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities.

PubMed

Schmidt, Béla Z; Lehmann, Martin; Gutbier, Simon; Nembo, Erastus; Noel, Sabrina; Smirnova, Lena; Forsby, Anna; Hescheler, Jürgen; Avci, Hasan X; Hartung, Thomas; Leist, Marcel; Kobolák, Julianna; Dinnyés, András

2017-01-01

Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.

PubMed

Kazuta, Yasuaki; Matsuura, Tomoaki; Ichihashi, Norikazu; Yomo, Tetsuya

2014-11-01

In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and β-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

PubMed

Thoma, Eva C; Heckel, Tobias; Keller, David; Giroud, Nicolas; Leonard, Brian; Christensen, Klaus; Roth, Adrian; Bertinetti-Lapatki, Cristina; Graf, Martin; Patsch, Christoph

2016-10-25

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Validation of in vitro assays in three-dimensional human dermal constructs.

PubMed

Idrees, Ayesha; Chiono, Valeria; Ciardelli, Gianluca; Shah, Siegfried; Viebahn, Richard; Zhang, Xiang; Salber, Jochen

2018-05-01

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially "murine in vitro dermal construct" based on L929 cells was generated, leading to the development of "human in vitro dermal construct" consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue ® , RealTime-Glo ™ MT, and CellTiter-Glo ® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the "shaking time" to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

[Spheroids: A reference model for in vitro culture of solid tumors?

PubMed

Larsen, Christian-Jacques

2018-01-01

The recognition that solid tumors are complex entities composed of the tumor cell mass itself and a stromal micro-environnement providing a variety of cells from the host (fibroblasts, endothelial cells, immune cells) led to recognize that this heterogeneity could not be recapitulated in vitro by conventional bidimensional (2-D) cultures. This justified numerous attempts to develop tridimensional (3-D) cultures that provided better tools for approaching tumor complexity and more convincing drug testing systems. Among various 3-D technologies, tumor spheroids are more likely suited to provide in vitro platforms for apprehending specific aspects of different processes specifically defining each tumor category as well as testing drug delivery systems. This review summarizes current features of multicellular tumor spheroids and their suitability for studying different aspects of cancer cell biology, patient-specific therapies and drug treatment. Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Analysis of the Material Properties of Early Chondrogenic Differentiated Adipose-Derived Stromal Cells (ASC) Using an in vitro Three-dimensional Micromass Culture System

PubMed Central

Xu, Yue; Balooch, Guive; Chiou, Michael; Bekerman, Elena; Ritchie, Robert O.; Longaker, Michael T.

2009-01-01

Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation. PMID:17543281

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

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

Rasey, J.S.; Nelson, N.J.

1983-01-01

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D/sub 0/more » of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.« less

Stem cell-derived systems in toxicology assessment.

PubMed

Suter-Dick, Laura; Alves, Paula M; Blaauboer, Bas J; Bremm, Klaus-Dieter; Brito, Catarina; Coecke, Sandra; Flick, Burkhard; Fowler, Paul; Hescheler, Jürgen; Ingelman-Sundberg, Magnus; Jennings, Paul; Kelm, Jens M; Manou, Irene; Mistry, Pratibha; Moretto, Angelo; Roth, Adrian; Stedman, Donald; van de Water, Bob; Beilmann, Mario

2015-06-01

Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.

“Do-it-yourself in vitro vasculature that recapitulates in vivo geometries for investigating endothelial-blood cell interactions”

PubMed Central

Mannino, Robert G.; Myers, David R.; Ahn, Byungwook; Wang, Yichen; Margo Rollins; Gole, Hope; Lin, Angela S.; Guldberg, Robert E.; Giddens, Don P.; Timmins, Lucas H.; Lam, Wilbur A.

2015-01-01

Investigating biophysical cellular interactions in the circulation currently requires choosing between in vivo models, which are difficult to interpret due in part to the hemodynamic and geometric complexities of the vasculature; or in vitro systems, which suffer from non-physiologic assumptions and/or require specialized microfabrication facilities and expertise. To bridge that gap, we developed an in vitro “do-it-yourself” perfusable vasculature model that recapitulates in vivo geometries, such as aneurysms, stenoses, and bifurcations, and supports endothelial cell culture. These inexpensive, disposable devices can be created rapidly (<2 hours) with high precision and repeatability, using standard off-the-shelf laboratory supplies. Using these “endothelialized” systems, we demonstrate that spatial variation in vascular cell adhesion molecule (VCAM-1) expression correlates with the wall shear stress patterns of vascular geometries. We further observe that the presence of endothelial cells in stenoses reduces platelet adhesion but increases sickle cell disease (SCD) red blood cell (RBC) adhesion in bifurcations. Overall, our method enables researchers from all disciplines to study cellular interactions in physiologically relevant, yet simple-to-make, in vitro vasculature models. PMID:26202603

Neuromuscular Junction Formation between Human Stem cell-derived Motoneurons and Human Skeletal Muscle in a Defined System

PubMed Central

Guo, Xiufang; Gonzalez, Mercedes; Stancescu, Maria; Vandenburgh, Herman; Hickman, James

2011-01-01

Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time lapse recordings and their subsequent quenching by D-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair. PMID:21944471

Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system.

PubMed

Guo, Xiufang; Gonzalez, Mercedes; Stancescu, Maria; Vandenburgh, Herman H; Hickman, James J

2011-12-01

Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time-lapse recordings and their subsequent quenching by d-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair. Copyright © 2011 Elsevier Ltd. All rights reserved.

An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

PubMed

Ruiz-Aracama, Ainhoa; Peijnenburg, Ad; Kleinjans, Jos; Jennen, Danyel; van Delft, Joost; Hellfrisch, Caroline; Lommen, Arjen

2011-05-20

In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively. The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD. Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.

In vitro fabrication of a tissue engineered human cardiovascular patch for future use in cardiovascular surgery.

PubMed

Yang, Chao; Sodian, Ralf; Fu, Ping; Lüders, Cora; Lemke, Thees; Du, Jing; Hübler, Michael; Weng, Yuguo; Meyer, Rudolf; Hetzer, Roland

2006-01-01

One approach to tissue engineering has been the development of in vitro conditions for the fabrication of functional cardiovascular structures intended for implantation. In this experiment, we developed a pulsatile flow system that provides biochemical and biomechanical signals in order to regulate autologous, human patch-tissue development in vitro. We constructed a biodegradable patch scaffold from porous poly-4-hydroxy-butyrate (P4HB; pore size 80 to 150 microm). The scaffold was seeded with pediatric aortic cells. The cell-seeded patch constructs were placed in a self-developed bioreactor for 7 days to observe potential tissue formation under dynamic cell culture conditions. As a control, cell-seeded scaffolds were not conditioned in the bioreactor system. After maturation in vitro, the analysis of the tissue engineered constructs included biochemical, biomechanical, morphologic, and immunohistochemical examination. Macroscopically, all tissue engineered constructs were covered by cells. After conditioning in the bioreactor, the cells were mostly viable, had grown into the pores, and had formed tissue on the patch construct. Electron microscopy showed confluent smooth surfaces. Additionally, we demonstrated the capacity to generate collagen and elastin under in vitro pulsatile flow conditions in biochemical examination. Biomechanical testing showed mechanical properties of the tissue engineered human patch tissue without any statistical differences in strength or resistance to stretch between the static controls and the conditioned patches. Immunohistochemical examination stained positive for alpha smooth muscle actin, collagen type I, and fibronectin. There was minor tissue formation in the nonconditioned control samples. Porous P4HB may be used to fabricate a biodegradable patch scaffold. Human vascular cells attached themselves to the polymeric scaffold, and extracellular matrix formation was induced under controlled biomechanical and biodynamic stimuli in a self-developed pulsatile bioreactor system.

Critical Evaluation of Air-Liquid Interface Cell Exposure Systems for In Vitro Assessment of Atmospheric Pollutants##

EPA Science Inventory

Conventional in vitro exposure studies of airborne pollutants involve, for example, the addition of particulate matter (PM) or PM extracts to the cell culture medium, or the bubbling of gases into the culture medium; these methods alter the pollutant’s physical and chemical...

Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3.

PubMed

De, B P; Galinski, M S; Banerjee, A K

1990-03-01

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

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

Taxvig, Camilla, E-mail: camta@food.dtu.dk; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenousmore » metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.« less

Moloney leukemia virus immortalizes B lymphocytes in vitro.

PubMed Central

Runnels, J; Serunian, L; Thursby, M; Rosenberg, N

1991-01-01

An in vitro culture system in which Moloney murine leukemia virus induces immortalization of mature B lymphocytes has been developed. The cell lines derived in this way are nontumorigenic, and virus production is not required to sustain them. This system provides a new in vitro model with which to study the stepwise process of transformation by retroviruses lacking oncogenes. Images PMID:1895405

Co-culture of stromal and erythroleukemia cells in a perfused hollow fiber bioreactor system as an in vitro bone marrow model for myeloid leukemia.

PubMed

Usuludin, Suaidah Binte Mohamed; Cao, Xue; Lim, Mayasari

2012-05-01

We have developed a hematopoietic co-culture system using the hollow fiber bioreactor (HFBR) as a potential in vitro bone marrow model for evaluating leukemia. Supporting stroma using HS-5 cells was established in HFBR system and the current bioprocess configuration yielded an average glucose consumption of 640 mg/day and an average protein concentration of 6.40 mg/mL in the extracapillary space over 28 days. Co-culture with erythroleukemia K562 cells was used as a model for myelo-leukemic cell proliferation and differentiation. Two distinct localizations of K562 cells (loosely adhered and adherent cells) were identified and characterized after 2 weeks. The HFBR co-culture resulted in greater leukemic cell expansion (3,130 fold vs. 43 fold) compared to a standard tissue culture polystyrene (TCP) culture. Majority of expanded cells (68%) in HFBR culture were the adherent population, highlighting the importance of cell-cell contact for myelo-leukemic proliferation. Differentiation tendencies in TCP favored maturation toward monocyte and erythrocyte lineages but maintained a pool of myeloid progenitors. In contrast, HFBR co-culture exhibited greater lineage diversity, stimulating monocytic and megakaryocytic differentiation while inhibiting erythroid maturation. With the extensive stromal expansion capacity on hollow fiber surfaces, the HFBR system is able to achieve high cell densities and 3D cell-cell contacts mimicking the bone marrow microenvironment. The proposed in vitro system represents a dynamic and highly scalable 3D co-culture platform for the study of cell-stroma dependent hematopoietic/leukemic cell functions and ex vivo expansion. Copyright © 2011 Wiley Periodicals, Inc.

Self-organization of neural tissue architectures from pluripotent stem cells.

PubMed

Karus, Michael; Blaess, Sandra; Brüstle, Oliver

2014-08-15

Despite being a subject of intensive research, the mechanisms underlying the formation of neural tissue architectures during development of the central nervous system remain largely enigmatic. So far, studies into neural pattern formation have been restricted mainly to animal experiments. With the advent of pluripotent stem cells it has become possible to explore early steps of nervous system development in vitro. These studies have unraveled a remarkable propensity of primitive neural cells to self-organize into primitive patterns such as neural tube-like rosettes in vitro. Data from more advanced 3D culture systems indicate that this intrinsic propensity for self-organization can even extend to the formation of complex architectures such as a multilayered cortical neuroepithelium or an entire optic cup. These novel experimental paradigms not only demonstrate the enormous self-organization capacity of neural stem cells, they also provide exciting prospects for studying the earliest steps of human neural tissue development and the pathogenesis of brain malformations in reductionist in vitro paradigms. © 2014 Wiley Periodicals, Inc.

Expression of genes responsible for cell morphogenesis involved in differentiation in porcine buccal pouch mucosal cells during long-term primary culture and real-time proliferation in vitro.

PubMed

Dyszkiewicz-Konwińska, M; Bryja, A; Jopek, K; Budna, J; Khozmi, R; Jeseta, M; Bukowska, D; Antosik, P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

2017-01-01

Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.

Allometric Scaling and Cell Ratios in Multi-Organ in vitro Models of Human Metabolism.

PubMed

Ucciferri, Nadia; Sbrana, Tommaso; Ahluwalia, Arti

2014-01-01

Intelligent in vitro models able to recapitulate the physiological interactions between tissues in the body have enormous potential as they enable detailed studies on specific two-way or higher order tissue communication. These models are the first step toward building an integrated picture of systemic metabolism and signaling in physiological or pathological conditions. However, the rational design of in vitro models of cell-cell or cell-tissue interaction is difficult as quite often cell culture experiments are driven by the device used, rather than by design considerations. Indeed, very little research has been carried out on in vitro models of metabolism connecting different cell or tissue types in a physiologically and metabolically relevant manner. Here, we analyze the physiological relationship between cells, cell metabolism, and exchange in the human body using allometric rules, downscaling them to an organ-on-a-plate device. In particular, in order to establish appropriate cell ratios in the system in a rational manner, two different allometric scaling models (cell number scaling model and metabolic and surface scaling model) are proposed and applied to a two compartment model of hepatic-vascular metabolic cross-talk. The theoretical scaling studies illustrate that the design and hence relevance of multi-organ models is principally determined by experimental constraints. Two experimentally feasible model configurations are then implemented in a multi-compartment organ-on-a-plate device. An analysis of the metabolic response of the two configurations demonstrates that their glucose and lipid balance is quite different, with only one of the two models recapitulating physiological-like homeostasis. In conclusion, not only do cross-talk and physical stimuli play an important role in in vitro models, but the numeric relationship between cells is also crucial to recreate in vitro interactions, which can be extrapolated to the in vivo reality.

Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro

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

Dubiak-Szepietowska, Monika, E-mail: Monika.Dubiak-Szepietowska@fh-jena.de; Karczmarczyk, Aleksandra; Jönsson-Niedziółka, Martin

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, we have developed and tested three dimensional environments for cells using different types of hydrogels including transglutaminase-cross-linked gelatin, collagen type I, and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, analysis of urea and albumin biosynthesis revealed that the created system allowed the immortalized liver cell line HepG2 to re-establish normal hepatocyte-like properties which weremore » not observed under the conditions of conventional cell cultures. This study presents a scalable technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates a higher cytotoxic effect when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that the cell environment has significant influence on cell sensitivity and that liver spheroid is a useful and novel tool to examine nanoparticle dosing effect even at the level of in vitro studies. Therefore, this system can be applied to a wide variety of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions. - Highlights: • Comparison of HepG2 cells growth in Matrigel, Collagen I gel and gelatin gel. • Examination of nanoparticles (NP) dosing effect at the level of in vitro studies. • Influence of the cell culture media composition on the cytotoxic effect of NP.« less

Cell-microenvironment interactions and architectures in microvascular systems

PubMed Central

Bersini, Simone; Yazdi, Iman K.; Talò, Giuseppe; Shin, Su Ryon; Moretti, Matteo; Khademhosseini, Ali

2016-01-01

In the past decade, significant advances have been made in the design and optimization of novel biomaterials and microfabrication techniques to generate vascularized tissues. Novel microfluidic systems have facilitated the development and optimization of in vitro models for exploring the complex pathophysiological phenomena that occur inside a microvascular environment. To date, most of these models have focused on engineering of increasingly complex systems, rather than analyzing the molecular and cellular mechanisms that drive microvascular network morphogenesis and remodeling. In fact, mutual interactions among endothelial cells (ECs), supporting mural cells and organ-specific cells, as well as between ECs and the extracellular matrix, are key driving forces for vascularization. This review focuses on the integration of materials science, microengineering and vascular biology for the development of in vitro microvascular systems. Various approaches currently being applied to study cell-cell/cell-matrix interactions, as well as biochemical/biophysical cues promoting vascularization and their impact on microvascular network formation, will be identified and discussed. Finally, this review will explore in vitro applications of microvascular systems, in vivo integration of transplanted vascularized tissues, and the important challenges for vascularization and controlling the microcirculatory system within the engineered tissues, especially for microfabrication approaches. It is likely that existing models and more complex models will further our understanding of the key elements of vascular network growth, stabilization and remodeling to translate basic research principles into functional, vascularized tissue constructs for regenerative medicine applications, drug screening and disease models. PMID:27417066

Cell-microenvironment interactions and architectures in microvascular systems.

PubMed

Bersini, Simone; Yazdi, Iman K; Talò, Giuseppe; Shin, Su Ryon; Moretti, Matteo; Khademhosseini, Ali

2016-11-01

In the past decade, significant advances have been made in the design and optimization of novel biomaterials and microfabrication techniques to generate vascularized tissues. Novel microfluidic systems have facilitated the development and optimization of in vitro models for exploring the complex pathophysiological phenomena that occur inside a microvascular environment. To date, most of these models have focused on engineering of increasingly complex systems, rather than analyzing the molecular and cellular mechanisms that drive microvascular network morphogenesis and remodeling. In fact, mutual interactions among endothelial cells (ECs), supporting mural cells and organ-specific cells, as well as between ECs and the extracellular matrix, are key driving forces for vascularization. This review focuses on the integration of materials science, microengineering and vascular biology for the development of in vitro microvascular systems. Various approaches currently being applied to study cell-cell/cell-matrix interactions, as well as biochemical/biophysical cues promoting vascularization and their impact on microvascular network formation, will be identified and discussed. Finally, this review will explore in vitro applications of microvascular systems, in vivo integration of transplanted vascularized tissues, and the important challenges for vascularization and controlling the microcirculatory system within the engineered tissues, especially for microfabrication approaches. It is likely that existing models and more complex models will further our understanding of the key elements of vascular network growth, stabilization and remodeling to translate basic research principles into functional, vascularized tissue constructs for regenerative medicine applications, drug screening and disease models. Copyright © 2016 Elsevier Inc. All rights reserved.

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro.

PubMed

Ishiwata, Isamu; Tamagawa, Tomoharu; Tokieda, Yuko; Iguchi, Megumi; Sato, Kahei; Ishikawa, Hiroshi

2003-03-01

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.

A non-laser light source for photodynamic therapy: in vitro effects on normal and malignant cells.

PubMed

Kashtan, Hanoch; Haddad, Riad; Greenberg, Ron; Skornick, Yehuda; Kaplan, Ofer

2002-01-01

Photodynamic therapy (PDT) involves the use of photosensitizing drugs combined with light to treat tumors. Laser systems, the current source of light for PDT, have several inherent drawbacks: the spectrum is essentially monochromatic which may be problematic for second generation photosensitizers, the systems are bulky and nearly impossible to move between hospital locations and require complicated electrical and cooling installations, the cost of a typical system is enormous, and its maintenance and operation require highly trained personnel. We now introduce a new non-laser light system, Versa-Light, which appears to work as effectively and has none of the above drawbacks. A series of in vitro studies were performed using various murine and human normal and cancer cells which underwent PDT using aluminum phthalocyanine (AlPcS4) as a photosensitizer and Versa-Light as the light source. PDT of cancer cells at light energy levels of 50, 100 and 200 j/cm2 significantly decreased cell viability. PDT also decreased cell viability of normal murine splenocytes and normal human lymphocytes, but to a lesser extent. The observed significant hyperthermia was light dose-dependent. We believe that Versa-Light can replace laser systems as an enhanced light source for PDT. Further in vitro and pre-clinical studies are in progress.

Photoacoustic detection of induced melanoma in vitro using a mouse model

NASA Astrophysics Data System (ADS)

Gupta, Sagar; Bhattacharya, Kiran; Newton, Jessica R.; Quinn, Thomas P.; Viator, John A.

2012-03-01

Metastasis is a life threatening complex physiological phenomenon that involves the movement of cancer cells from one organ to another by means of blood and lymph. An understanding about metastasis is extremely important to device diagnostic systems to detect and monitor its spread within the body. For the first time we report rapid photoacoustic detection of the induced metastatic melanoma in mice in vitro using photoacoustic flowmetry. A new photoacoustic flow system is developed, that employs photoacoustic excitation coupled with an ultrasound transducer capable of determining the presence of individual, induced mouse melanoma cells (B16/F10) within the circulating system in vitro. Tumor was induced in mice by injecting mouse melanoma cells through tail vein into the C57BL/6 mice. A luciferase based in vivo bioluminescence imaging is performed to confirm the tumor load and multiple metastases in the tumor-induced mice. 1ml of blood obtained through cardiac puncture of the induced metastasized mice was treated to lyse the red blood cells (RBC) and enriched, leaving the induced melanoma in the peripheral blood mononuclear suspension (PBMC). A photoacoustic flowsystem coupled with an ultrasound transducer is used to detect the individual circulating metastatic melanoma cells from the enriched cell suspension.

In vitro immunomodulatory potential of Artemisia indica Willd. in chicken lymphocytes.

PubMed

Ruwali, Pushpa; Ambwani, Tanuj Kumar; Gautam, Pankaj

2018-01-01

Evaluation of the in vitro immunomodulatory potential of Artemisia indica Willd. methanolic extract in chicken lymphocyte culture system through lymphocyte (B and T cells) proliferation assay, after standardizing the maximum non-cytotoxic dose (MNCD) in chicken lymphocytes. Fresh aerial parts of A. indica Willd. (family: Asteraceae) specimens were collected (altitude 1560 m), gotten authenticated, processed, dried, and Soxhlet extracted to yield methanolic extract (AME). Chicken splenocytes were isolated from spleens collected from healthy birds; lymphocytes were separated by density gradient centrifugation, percentage cell viability determined and final cell count adjusted to 10 7 cells/ml in RPMI-1640 medium. MNCD of AME in chicken lymphocytes was determined through 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide dye reduction assay. Immunomodulatory potential of AME was evaluated through lymphocytes proliferation or B and T cells blastogenesis assay in the presence of appropriate mitogens, namely, lipopolysaccharide (LPS) and concanavalin A (Con A), respectively. Maximum concentration of AME exhibiting 100% cell viability (MNCD) was 200 μg/ml and was selected for further in vitro analysis. The in vitro exposure of chicken lymphocytes to 200 µg/ml dose of AME, resulted in significant (p<0.05) upregulation of 11.76% in B cell proliferation in the presence of B cell mitogen (LPS) and a significant (p<0.05) increase of 12.018% T cells proliferation in the presence of the mitogen (Con A), as compared to the control. The significant upregulation in the proliferation of two major cell types modulating the immune system is an indication of the immunostimulatory potential of the plant. It would be worthwhile to further evaluate A. indica on relevant immunomodulatory aspects, especially the in vivo studies in a poultry system.

Engineering of mesoporous silica nanoparticles for release of ginsenoside CK and Rh2 to enhance their anticancer and anti-inflammatory efficacy: in vitro studies

NASA Astrophysics Data System (ADS)

Singh, Priyanka; Singh, Hina; Castro-Aceituno, Verónica; Ahn, Sungeun; Kim, Yeon Ju; Farh, Mohamed El-Agamy; Yang, Deok Chun

2017-07-01

The current study highlights the fabrication of drug delivery system by utilizing 200 nm mesoporous silica nanoparticles (MSNPs) with 4-nm pore size, as a carrier system for delivery ginsenoside compound K (CK) and Rh2 to enhance their efficacy. The two pharmacologically imperative ginsenosides, CK and Rh2, were loaded to the MSNPs to prepare MSNPs-CK and MSNPs-Rh2, respectively. A fluorescein isothiocyanate (FITC) fluorescent dye was combined in the MSNPs carrier system, in order to trace the cellular uptake of ginsenoside-loaded nanoparticles for in vitro studies. Following purification, the so-prepared MSNPs-CK-FITC and MSNPs-Rh2-FITC were characterized by several analytical techniques, which includes, high-pressure liquid chromatography (HPLC), 1H NMR, field emission transmission electron microscopy (FE-TEM), Fourier transform infrared spectroscopy (FT-IR), x-ray diffraction (XRD), thermogravimetric analysis (TGA), and dynamic light scattering (DLS). In vitro cytotoxicity assay in HaCaT skin cells, A549 lung cancer cells, HepG2 liver carcinoma cells, and HT-29 colon cancer cell lines were tested for MSNPs-CK-FITC and MSNPs-Rh2-FITC. The results demonstrate the excellent biocompatibility of nanoparticles in normal cell lines (HaCaT skin cells) and anticancer efficacy in all the tested cancer cell lines at 10-μM concentration. Additionally, the in vitro anti-inflammatory behavior of MSNPs-CK-FITC and MSNPs-Rh2-FITC were checked in RAW264.7 (murine macrophage) cell lines. The outcomes showed higher anti-inflammatory efficacy of MSNPs-CK-FITC and MSNPs-Rh2-FITC as compared to standard ginsenosides CK and Rh2 in RAW264.7 cell lines. Thus, with 200 nm MSNPs carrier system for the delivery ginsenosides CK and Rh2, a high amount of loading and increasing in vitro pharmacological efficacies of ginsenosides were realized. This study may provide useful insights for designing and improving the applicability of MSNPs for ginsenoside delivery.

Cell chirality: emergence of asymmetry from cell culture.

PubMed

Wan, Leo Q; Chin, Amanda S; Worley, Kathryn E; Ray, Poulomi

2016-12-19

Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left-right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

Cell chirality: emergence of asymmetry from cell culture

PubMed Central

Wan, Leo Q.; Chin, Amanda S.; Worley, Kathryn E.; Ray, Poulomi

2016-01-01

Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left–right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821525

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

PubMed

Chiba, Eriko; Villena, Julio; Hosoya, Shoichi; Takanashi, Naoya; Shimazu, Tomoyuki; Aso, Hisashi; Tohno, Masanori; Suda, Yoshihito; Kawai, Yasushi; Saito, Tadao; Miyazawa, Kenji; He, Fang; Kitazawa, Haruki

2012-10-01

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts. Copyright © 2011 Elsevier Ltd. All rights reserved.

Classification of phthalates based on an in vitro neurosphere assay using rat mesencephalic neural stem cells.

PubMed

Ishido, Masami; Suzuki, Junko

2014-02-01

Exposure to environmental neurotoxic chemicals both in utero and during the early postnatal period can cause neurodevelopmental disorders. To evaluate the disruption of neurodevelopmental programming, we previously established an in vitro neurosphere assay system using rat mesencephalic neural stem cells that can be used to evaluate. Here, we extended the assay system to examine the neurodevelopmental toxicity of the endocrine disruptors butyl benzyl phthalate, di-n-butyl phthalate, dicyclohexyl phthalate, diethyl phthalate, di(2-ethyl hexyl) phthalate, di-n-pentyl phthalate, and dihexyl phthalate at a range of concentrations (0-100 μM). All phthalates tested inhibited cell migration with a linear or non-linear range of concentrations when comparing migration distance to the logarithm of the phthalate concentrations. On the other hand, some, but not all, phthalates decreased the number of proliferating cells. Apoptotic cells were not observed upon phthalate exposure under any of the conditions tested, whereas the dopaminergic toxin rotenone induced significant apoptosis. Thus, we were able to classify phthalate toxicity based on cell migration and cell proliferation using the in vitro neurosphere assay.

40 CFR 798.5375 - In vitro mammalian cytogenetics.

Code of Federal Regulations, 2014 CFR

2014-07-01

... mammalian cytogenetics. (a) Purpose. The in vitro cytogenetics test is a mutagenicity test system for the... first post-treatment mitosis and numerical aberrations require at least one cell division to be... chromatids. (c) Reference substances. Not applicable. (d) Test method—(1) Principle. In vitro cytogenetics...

Mammalian cell display technology coupling with AID induced SHM in vitro: an ideal approach to the production of therapeutic antibodies.

PubMed

Qin, Chang-Fei; Li, Guan-Cheng

2014-12-01

Traditional antibody production technology within non-mammalian cell expression systems has shown many unsatisfactory properties for the development of therapeutic antibodies. Nevertheless, mammalian cell display technology reaps the benefits of producing full-length all human antibodies. Together with the developed cytidine deaminase induced in vitro somatic hypermutation technology, mammalian cell display technology provides the opportunity to produce high affinity antibodies that might be ideal for therapeutic application. This review was concentrated on the development of the mammalian cell display technology as well as the activation-induced cytidine deaminase induced in vitro somatic hypermutation technology and their applications for the production of therapeutic antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay.

PubMed

Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects. Copyright © 2010 Elsevier Inc. All rights reserved.

Allometric Scaling and Cell Ratios in Multi-Organ in vitro Models of Human Metabolism

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

Ucciferri, Nadia; Interdepartmental Research Center “E. Piaggio”, University of Pisa, Pisa; Sbrana, Tommaso

2014-12-17

Intelligent in vitro models able to recapitulate the physiological interactions between tissues in the body have enormous potential as they enable detailed studies on specific two-way or higher order tissue communication. These models are the first step toward building an integrated picture of systemic metabolism and signaling in physiological or pathological conditions. However, the rational design of in vitro models of cell–cell or cell–tissue interaction is difficult as quite often cell culture experiments are driven by the device used, rather than by design considerations. Indeed, very little research has been carried out on in vitro models of metabolism connecting differentmore » cell or tissue types in a physiologically and metabolically relevant manner. Here, we analyze the physiological relationship between cells, cell metabolism, and exchange in the human body using allometric rules, downscaling them to an organ-on-a-plate device. In particular, in order to establish appropriate cell ratios in the system in a rational manner, two different allometric scaling models (cell number scaling model and metabolic and surface scaling model) are proposed and applied to a two compartment model of hepatic-vascular metabolic cross-talk. The theoretical scaling studies illustrate that the design and hence relevance of multi-organ models is principally determined by experimental constraints. Two experimentally feasible model configurations are then implemented in a multi-compartment organ-on-a-plate device. An analysis of the metabolic response of the two configurations demonstrates that their glucose and lipid balance is quite different, with only one of the two models recapitulating physiological-like homeostasis. In conclusion, not only do cross-talk and physical stimuli play an important role in in vitro models, but the numeric relationship between cells is also crucial to recreate in vitro interactions, which can be extrapolated to the in vivo reality.« less

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

Maneckjee, R.; Minna, J.D.

Using specific radioactively-labeled ligands, the authors find that lung cancer cell lines of diverse histologic types express multiple, high-affinity membrane receptors for {mu}, {delta}, and {kappa} opioid agonists and for nicotine and {alpha}-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas {mu}, {delta}, and {kappa} opioid agonists at low concentrations inhibited lung cancer growth in vitro. They also found that lung cancer cells expressed various combinations of immunoreactive opioid peptidesmore » ({beta}-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, they tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a tumor suppressor system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer.« less

DynaMiTES - A dynamic cell culture platform for in vitro drug testing PART 1 - Engineering of microfluidic system and technical simulations.

PubMed

Mattern, Kai; Beißner, Nicole; Reichl, Stephan; Dietzel, Andreas

2018-05-01

Conventional safety and efficacy test models, such as animal experiments or static in vitro cell culture models, can often not reliably predict the most promising drug candidates. Therefore, a novel microfluidic cell culture platform, called Dynamic Micro Tissue Engineering System (DynaMiTES), was designed to allow online analysis of drugs permeating through barrier forming tissues under dynamic conditions combined with monitoring of the transepithelial electrical resistance (TEER) by electrodes optimized for homogeneous current distribution. A variety of pre-cultivated cell culture inserts can be integrated and exposed to well controlled dynamic micro flow conditions, resulting in a tightly regulated exposure of the cells to tested drugs, drug formulations and shear forces. With these qualities, the new system can provide more relevant information compared to static measurements. As a first in vitro model, a three-dimensional hemicornea construct consisting of human keratocytes (HCK-Ca) and epithelial cells (HCE-T) was successfully tested in the DynaMiTES. Thereby, we were able to demonstrate the functionality and cell compatibility of this new organ on chip test platform. The modular design of the DynaMiTES allows fast adaptation suitable for the investigation of drug permeation through other important cellular barriers. Copyright © 2017. Published by Elsevier B.V.

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

PubMed

Rozov, F N; Grinenko, T S; Levit, G L; Krasnov, V P; Belyavsky, A V

2010-09-15

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders. 2010 Elsevier Inc. All rights reserved.

Development of a microfluidic perfusion 3D cell culture system

NASA Astrophysics Data System (ADS)

Park, D. H.; Jeon, H. J.; Kim, M. J.; Nguyen, X. D.; Morten, K.; Go, J. S.

2018-04-01

Recently, 3-dimensional in vitro cell cultures have gained much attention in biomedical sciences because of the closer relevance between in vitro cell cultures and in vivo environments. This paper presents a microfluidic perfusion 3D cell culture system with consistent control of long-term culture conditions to mimic an in vivo microenvironment. It consists of two sudden expansion reservoirs to trap incoming air bubbles, gradient generators to provide a linear concentration, and microchannel mixers. Specifically, the air bubbles disturb a flow in the microfluidic channel resulting in the instability of the perfusion cell culture conditions. For long-term stable operation, the sudden expansion reservoir is designed to trap air bubbles by using buoyancy before they enter the culture system. The performance of the developed microfluidic perfusion 3D cell culture system was examined experimentally and compared with analytical results. Finally, it was applied to test the cytotoxicity of cells infected with Ewing’s sarcoma. Cell death was observed for different concentrations of H2O2. For future work, the developed microfluidic perfusion 3D cell culture system can be used to examine the behavior of cells treated with various drugs and concentrations for high-throughput drug screening.

Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

PubMed Central

Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

2015-01-01

Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

The bovine placenta in vivo and in vitro.

PubMed

Haeger, J-D; Hambruch, N; Pfarrer, C

2016-07-01

The gross anatomic features (cotyledonary type) and histologic classification (synepitheliochorial) of the bovine placenta have been known for many years. Thorough ultrastructural analysis as well as a variety of descriptive studies dealing with the localization of cytoskeletal filaments, extracellular matrix, growth factor systems, steroid hormone receptors, and major histocompatibility complex have contributed further significant knowledge. However, this knowledge was not sufficient to solve clinical placenta-based problems, such as retained fetal membranes. Owing to the complexity of the fetomaternal interface in vitro, culture systems have been developed. As trophoblast giant cells (TGC) are thought to be key players in the cattle placenta, most cell culture models attempt to overcome the pitfall of losing the entire TGC population in vitro. Nevertheless, distinct cell line-based in vitro systems such as cell monolayers or 3-dimensional (co-culture) spheroids were generated for the fetal (trophoblast) and maternal (uterine epithelium) placental compartments. Monolayers have been used to study for example, growth factor or hormonal signaling and TGC formation, whereas spheroids served as models for, for example, trophoblast attachment, uterine epithelium depolarization, and also TGC formation. In the future, the use of more improved culture models might lead to better treatments of retained fetal membranes and increased prevention of embryonic loss. In addition, the in vitro models could shed more light on the mechanisms of the differentiation of uninucleate trophoblast into TGC. Copyright © 2016 Elsevier Inc. All rights reserved.

ISD3: a particokinetic model for predicting the combined effects of particle sedimentation, diffusion and dissolution on cellular dosimetry for in vitro systems

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

Thomas, Dennis G.; Smith, Jordan N.; Thrall, Brian D.

The development of particokinetic models describing the delivery of insoluble or poorly soluble nanoparticles to cells in liquid cell culture systems has improved the basis for dose-response analysis, hazard ranking from high-throughput systems, and now allows for translation of exposures across in vitro and in vivo test systems. Complimentary particokinetic models that address processes controlling delivery of both particles and released ions to cells, and the influence of particle size changes from dissolution on particle delivery for cell-culture systems would help advance our understanding of the role of particles ion dosimetry on cellular toxicology. We developed ISD3, an extension ofmore » our previously published model for insoluble particles, by deriving a specific formulation of the Population Balance Equation for soluble particles. ISD3 describes the time, concentration and particle size dependent dissolution of particles, their delivery to cells, and the delivery and uptake of ions to cells in in vitro liquid test systems. The model is modular, and can be adapted by application of any empirical model of dissolution, alternative approaches to calculating sedimentation rates, and cellular uptake or treatment of boundary conditions. We apply the model to calculate the particle and ion dosimetry of nanosilver and silver ions in vitro after calibration of two empirical models, one for particle dissolution and one for ion uptake. The results demonstrate utility and accuracy of the ISD3 framework for dosimetry in these systems. Total media ion concentration, particle concentration and total cell-associated silver time-courses were well described by the model, across 2 concentrations of 20 and 110 nm particles. ISD3 was calibrated to dissolution data for 20 nm particles as a function of serum protein concentration, but successfully described the media and cell dosimetry time-course for both particles at all concentrations and time points. We also report the finding that protein content in media has effects both on the initial rate of dissolution and the resulting near-steady state ion concentration in solution.« less

Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway

PubMed Central

Leung, E; Xue, A; Wang, Y; Rougerie, P; Sharma, V P; Eddy, R; Cox, D; Condeelis, J

2017-01-01

During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming, where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing extracellular matrix (ECM) fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages and endothelial cells on 2.5 μm thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells were placed at one end of the micro-patterned ‘ECM fibers' within the assay. We demonstrated that these endothelial cells supply the hepatocyte growth factor (HGF) required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked, by inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel-directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo, and for use in human trials. PMID:27893712

Electrochemical microsensor system for cancer research on photodynamic therapy in vitro

NASA Astrophysics Data System (ADS)

Marzioch, J.; Kieninger, J.; Sandvik, J. A.; Pettersen, E. O.; Peng, Q.; Urban, G.

2016-10-01

An electrochemical microsensor system to investigate photodynamic therapy of cancer cells in vitro was developed and applied to monitor the cellular respiration during and after photodynamic therapy. The redox activity and therefore influence of the photodynamic drug on the sensor performance was investigated by electrochemical characterization. It was shown, that appropriate operation conditions avoid cross-sensitivity of the sensors to the drug itself. The presented system features a cell culture chamber equipped with microsensors and a laser source to photodynamically treat the cells while simultaneous monitoring of metabolic parameter in situ. Additionally, the optical setup allows to read back fluorescence signals from the photosensitizer itself or other marker molecules parallel to the microsensor readings.

Comparison of in vitro and in vivo effects of vincristine and vindesine on leukemic cells from patients with chronic granulocytic leukemia in blast crisis.

PubMed

Theodorakis, M E; Goldberg, J

1984-01-01

We employed a liquid culture system to examine the in vitro effects of vincristine and vindesine on cellular incorporation of 35SO4 into leukemic cells obtained from 5 patients with chronic granulocytic leukemia in blast crisis. The per cent of 35SO4 into drug-treated as compared to saline-treated leukemic cells was compared to the clinical outcome of patients treated with these agents. A good or partial clinical response to vincristine or vindesine was seen in patients whose leukemic cells incorporated less than 50% 35SO4 when exposed to vincristine or vindesine in vitro, compared with control saline-treated cells. No clinical response was observed following treatment with vincristine or vindesine if the 35SO4 incorporation of drug treated leukemic cells was greater than 50% of saline-treated cells. These data suggest that the in vitro effects of vincristine or vindesine on 35SO4 incorporation into leukemic cells of patients in blast crisis may parallel the clinical outcome of patients treated with these agents in vivo.

AN EVALUATION OF THE RELATIVE GENOTOXICITY OF ARSENITE, ARSENATE, AND FOUR METHYLATED METABOLITES IN VITRO USING THE ALKALINE SINGLE CELL GEL ASSAY

EPA Science Inventory

An Evaluation of the Relative Genotoxicity of Arsenite, Arsenate, and Four Methylated
Metabolites In Vitro Using the Alkaline Single Cell Gel Assay (ASCG).

Arsenic ( As) is a genotoxic and carcinogenic metal found in many drinking water systems throughout the world. ...

The current limitations of in vitro genotoxicity testing and their relevance to the in vivo situation.

PubMed

Nesslany, Fabrice

2017-08-01

The standard regulatory core battery of genotoxicity tests generally includes 2 or 3 validated tests with at least one in vitro test in bacteria and one in vitro test on cell cultures. However, limitations in in vitro genotoxicity testing may exist at many levels. The knowledge of the underlying mechanisms of genotoxicity is particularly useful to assess the level of relevance for the in vivo situation. In order to avoid wrong conclusions regarding the actual genotoxicity status of any test substance, it appears very important to be aware of the various origins of related bias leading to 'false positives and negatives' by using in vitro methods. Among these, mention may be made on the metabolic activation system, experimental (extreme) conditions, specificities of the test systems implemented, cell type used etc. The knowledge of the actual 'limits' of the in vitro test systems used is clearly an advantage and may contribute to avoid some pitfalls in order to better assess the level of relevance for the in vivo situation. Copyright © 2016. Published by Elsevier Ltd.

Reconstitution of mouse oogenesis in a dish from pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Hikabe, Orie; Obata, Yayoi; Hirao, Yuji

2017-09-01

This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

On-line observation of cell growth in a three-dimensional matrix on surface-modified microelectrode arrays.

PubMed

Lin, Shu-Ping; Kyriakides, Themis R; Chen, Jia-Jin J

2009-06-01

Despite many successful applications of microelectrode arrays (MEAs), typical two-dimensional in-vitro cultures do not project the full scale of the cell growth environment in the three-dimensional (3D) in-vivo setting. This study aims to on-line monitor in-vitro cell growth in a 3D matrix on the surface-modified MEAs with a dynamic perfusion culture system. A 3D matrix consisting of poly(ethylene glycol) hydrogel supplemented with poly-D-lysine was subsequently synthesized in situ on the self-assembled monolayer modified MEAs. FTIR spectrum analysis revealed a peak at 2100 cm(-1) due to the degradation of the structure of the 3D matrix. After 2 wks, microscopic examination revealed that the non-degraded area was around 1500 microm(2) and provided enough space for cell growth. Fluorescence microscopy revealed that the degraded 3D matrix was non-cytotoxic allowing the growth of NIH3T3 fibroblasts and cortical neurons in vitro. Time-course changes of total impedance including resistance and reactance were recorded for 8 days to evaluate the cell growth in the 3D matrix on the MEA. A consistent trend reflecting changes of reactance and total impedance was observed. These in-vitro assays demonstrate that our 3D matrix can construct a biomimetic system for cell growth and analysis of cell surface interactions.

Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.

PubMed

Kelwick, Richard; Webb, Alexander J; MacDonald, James T; Freemont, Paul S

2016-11-01

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P grac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

Hanging drop: an in vitro air toxic exposure model using human lung cells in 2D and 3D structures.

PubMed

Liu, Faye F; Peng, Cheng; Escher, Beate I; Fantino, Emmanuelle; Giles, Cindy; Were, Stephen; Duffy, Lesley; Ng, Jack C

2013-10-15

Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX(®) system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50cell for 1h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15 mmol/kgdry weight, which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method. Copyright © 2013 Elsevier B.V. All rights reserved.

Infection Spread and Virus Release in Vitro in Cell Populations as a System with Percolation

NASA Astrophysics Data System (ADS)

Ochoa, Juan G. Diaz

The comprehension of the innate immune system of cell populations is not only of interest to understand systems in vivo but also in vitro, for example, in the control of the release of viral particles for the production of vaccines. In this report I introduce a model, based on dynamical networks, that simulates the cell signaling responsible for this innate immune response and its effect on the infection spread and virus production. The central motivation is to represent a cell population that is constantly mixed in a bio-reactor where there is a cell-to-cell signaling of cytokines (which are proteins responsible for the activation of the antiviral response inside the cell). Such signaling allows the definition of clusters of linked immune cells. Additionally, depending on the density of links, it is possible to identify critical threshold parameters associated to a percolation phase transition. I show that the control of this antiviral response is equivalent to a percolation process.

Electroinduced Delivery of Hydrogel Nanoparticles in Colon 26 Cells, Visualized by Confocal Fluorescence System.

PubMed

Atanasova, Severina; Nikolova, Biliana; Murayama, Shuhei; Stoyanova, Elena; Tsoneva, Iana; Zhelev, Zhivko; Aoki, Ichio; Bakalova, Rumiana

2016-09-01

Nano-scale drug delivery systems (nano-DDS) are under intense investigation. Nano-platforms are developed for specific administration of small molecules, drugs, genes, contrast agents [quantum dots (QDs)] both in vivo and in vitro. Electroporation is a biophysical phenomenon which consists of the application of external electrical pulses across the cell membrane. The aim of this study was to research electro-assisted Colon 26 cell line internalization of QDs and QD-loaded nano-hydrogels (polymersomes) visualized by confocal microscopy and their influence on cell viability. The experiments were performed on the Colon 26 cancer cell line, using a confocal fluorescent imaging system and cell viability test. Electroporation facilitated the delivery of nanoparticles in vivo. We demonstrated increased voltage-dependent delivery of nanoparticles into cells after electrotreatment, without significant cell viability reduction. The delivery and retention of the polymersomes in vitro is a promising tool for future cancer treatment strategies and nanomedcine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Mammalian oocyte growth and development in vitro.

PubMed

Eppig, J J; O'Brien, M; Wigglesworth, K

1996-06-01

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes form preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk.

Self-renewal and multilineage differentiation of mouse dental epithelial stem cells.

PubMed

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L; D'Souza, Rena N; Wang, Fen

2013-11-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. © 2013.

Self-renewal and Multilineage Differentiation of Mouse Dental Epithelial Stem Cells

PubMed Central

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L.; D’Souza, Rena N.; Wang, Fen

2013-01-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49fBright population was enriched in sphere-forming cells. In addition, the CD49fBright population includes both slow-cycling and Lgr5+ DESCs. The in vitro sphere culture system and identification of CD49fBright as a DESC marker provide a novel plateform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. PMID:23906788

Invertebrate hematopoiesis: an anterior proliferation center as a link between the hematopoietic tissue and the brain.

PubMed

Noonin, Chadanat; Lin, Xionghui; Jiravanichpaisal, Pikul; Söderhäll, Kenneth; Söderhäll, Irene

2012-11-20

During evolution, the innate and adaptive immune systems were developed to protect organisms from non-self substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods lack the blood cells of the lymphoid lineage and oxygen-carrying erythrocytes, making them suitable model animals for studying the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model for studying hematopoiesis, because the tissue can be isolated, and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here, we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation center (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system, thereby obtaining new information about the evolution of different blood and nerve cell lineages.

Interferon-induced 2'-5' adenylate synthetase in vivo and interferon production in vitro by lymphocytes from systemic lupus erythematosus patients with and without circulating interferon

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

Preble, O.T.; Rothko, K.; Klippel, J.H.

1983-06-01

The interferon (IFN)-induced enzyme 2-5A synthetase was elevated in mononuclear cells from both serum IFN-positive and -negative systemic lupus erythematosus (SLE) patients. This suggests that a much higher percentage of patients than previously thought produce endogenous IFN. These results may partly explain findings that mononuclear cells from SLE patients are deficient in IFN production in vitro in response to certain IFN inducers. Although normal lymphocytes can produce an acid-labile alpha IFN after stimulation with C. parvum in vitro, the reason for endogenous production of this unusual alpha IFN by SLE patients remains unknown.

Measured and Modeled Toxicokinetics in Cultured Fish Cells and Application to In Vitro - In Vivo Toxicity Extrapolation

PubMed Central

Stadnicka-Michalak, Julita; Tanneberger, Katrin; Schirmer, Kristin; Ashauer, Roman

2014-01-01

Effect concentrations in the toxicity assessment of chemicals with fish and fish cells are generally based on external exposure concentrations. External concentrations as dose metrics, may, however, hamper interpretation and extrapolation of toxicological effects because it is the internal concentration that gives rise to the biological effective dose. Thus, we need to understand the relationship between the external and internal concentrations of chemicals. The objectives of this study were to: (i) elucidate the time-course of the concentration of chemicals with a wide range of physicochemical properties in the compartments of an in vitro test system, (ii) derive a predictive model for toxicokinetics in the in vitro test system, (iii) test the hypothesis that internal effect concentrations in fish (in vivo) and fish cell lines (in vitro) correlate, and (iv) develop a quantitative in vitro to in vivo toxicity extrapolation method for fish acute toxicity. To achieve these goals, time-dependent amounts of organic chemicals were measured in medium, cells (RTgill-W1) and the plastic of exposure wells. Then, the relation between uptake, elimination rate constants, and log KOW was investigated for cells in order to develop a toxicokinetic model. This model was used to predict internal effect concentrations in cells, which were compared with internal effect concentrations in fish gills predicted by a Physiologically Based Toxicokinetic model. Our model could predict concentrations of non-volatile organic chemicals with log KOW between 0.5 and 7 in cells. The correlation of the log ratio of internal effect concentrations in fish gills and the fish gill cell line with the log KOW was significant (r>0.85, p = 0.0008, F-test). This ratio can be predicted from the log KOW of the chemical (77% of variance explained), comprising a promising model to predict lethal effects on fish based on in vitro data. PMID:24647349

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.

Target specific delivery of anticancer drug in silk fibroin based 3D distribution model of bone-breast cancer cells.

PubMed

Subia, Bano; Dey, Tuli; Sharma, Shaily; Kundu, Subhas C

2015-02-04

To avoid the indiscriminating action of anticancer drugs, the cancer cell specific targeting of drug molecule becomes a preferred choice for the treatment. The successful screening of the drug molecules in 2D culture system requires further validation. The failure of target specific drug in animal model raises the issue of creating a platform in between the in vitro (2D) and in vivo animal testing. The metastatic breast cancer cells migrate and settle at different sites such as bone tissue. This work evaluates the in vitro 3D model of the breast cancer and bone cells to understand the cellular interactions in the presence of a targeted anticancer drug delivery system. The silk fibroin based cytocompatible 3D scaffold is used as in vitro 3D distribution model. Human breast adenocarcinoma and osteoblast like cells are cocultured to evaluate the efficiency of doxorubicin loaded folic acid conjugated silk fibroin nanoparticle as drug delivery system. Decreasing population of the cancer cells, which lower the levels of vascular endothelial growth factors, glucose consumption, and lactate production are observed in the drug treated coculture constructs. The drug treated constructs do not show any major impact on bone mineralization. The diminished expression of osteogenic markers such as osteocalcein and alkaline phosphatase are recorded. The result indicates that this type of silk based 3D in vitro coculture model may be utilized as a bridge between the traditional 2D and animal model system to evaluate the new drug molecule (s) or to reassay the known drug molecules or to develop target specific drug in cancer research.

ISD3: a particokinetic model for predicting the combined effects of particle sedimentation, diffusion and dissolution on cellular dosimetry for in vitro systems.

PubMed

Thomas, Dennis G; Smith, Jordan N; Thrall, Brian D; Baer, Donald R; Jolley, Hadley; Munusamy, Prabhakaran; Kodali, Vamsi; Demokritou, Philip; Cohen, Joel; Teeguarden, Justin G

2018-01-25

The development of particokinetic models describing the delivery of insoluble or poorly soluble nanoparticles to cells in liquid cell culture systems has improved the basis for dose-response analysis, hazard ranking from high-throughput systems, and now allows for translation of exposures across in vitro and in vivo test systems. Complimentary particokinetic models that address processes controlling delivery of both particles and released ions to cells, and the influence of particle size changes from dissolution on particle delivery for cell-culture systems would help advance our understanding of the role of particles and ion dosimetry on cellular toxicology. We developed ISD3, an extension of our previously published model for insoluble particles, by deriving a specific formulation of the Population Balance Equation for soluble particles. ISD3 describes the time, concentration and particle size dependent dissolution of particles, their delivery to cells, and the delivery and uptake of ions to cells in in vitro liquid test systems. We applied the model to calculate the particle and ion dosimetry of nanosilver and silver ions in vitro after calibration of two empirical models, one for particle dissolution and one for ion uptake. Total media ion concentration, particle concentration and total cell-associated silver time-courses were well described by the model, across 2 concentrations of 20 and 110 nm particles. ISD3 was calibrated to dissolution data for 20 nm particles as a function of serum protein concentration, but successfully described the media and cell dosimetry time-course for both particles at all concentrations and time points. We also report the finding that protein content in media affects the initial rate of dissolution and the resulting near-steady state ion concentration in solution for the systems we have studied. By combining experiments and modeling, we were able to quantify the influence of proteins on silver particle solubility, determine the relative amounts of silver ions and particles in exposed cells, and demonstrate the influence of particle size changes resulting from dissolution on particle delivery to cells in culture. ISD3 is modular and can be adapted to new applications by replacing descriptions of dissolution, sedimentation and boundary conditions with those appropriate for particles other than silver.

ISDD: A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies

PubMed Central

2010-01-01

Background The difficulty of directly measuring cellular dose is a significant obstacle to application of target tissue dosimetry for nanoparticle and microparticle toxicity assessment, particularly for in vitro systems. As a consequence, the target tissue paradigm for dosimetry and hazard assessment of nanoparticles has largely been ignored in favor of using metrics of exposure (e.g. μg particle/mL culture medium, particle surface area/mL, particle number/mL). We have developed a computational model of solution particokinetics (sedimentation, diffusion) and dosimetry for non-interacting spherical particles and their agglomerates in monolayer cell culture systems. Particle transport to cells is calculated by simultaneous solution of Stokes Law (sedimentation) and the Stokes-Einstein equation (diffusion). Results The In vitro Sedimentation, Diffusion and Dosimetry model (ISDD) was tested against measured transport rates or cellular doses for multiple sizes of polystyrene spheres (20-1100 nm), 35 nm amorphous silica, and large agglomerates of 30 nm iron oxide particles. Overall, without adjusting any parameters, model predicted cellular doses were in close agreement with the experimental data, differing from as little as 5% to as much as three-fold, but in most cases approximately two-fold, within the limits of the accuracy of the measurement systems. Applying the model, we generalize the effects of particle size, particle density, agglomeration state and agglomerate characteristics on target cell dosimetry in vitro. Conclusions Our results confirm our hypothesis that for liquid-based in vitro systems, the dose-rates and target cell doses for all particles are not equal; they can vary significantly, in direct contrast to the assumption of dose-equivalency implicit in the use of mass-based media concentrations as metrics of exposure for dose-response assessment. The difference between equivalent nominal media concentration exposures on a μg/mL basis and target cell doses on a particle surface area or number basis can be as high as three to six orders of magnitude. As a consequence, in vitro hazard assessments utilizing mass-based exposure metrics have inherently high errors where particle number or surface areas target cells doses are believed to drive response. The gold standard for particle dosimetry for in vitro nanotoxicology studies should be direct experimental measurement of the cellular content of the studied particle. However, where such measurements are impractical, unfeasible, and before such measurements become common, particle dosimetry models such as ISDD provide a valuable, immediately useful alternative, and eventually, an adjunct to such measurements. PMID:21118529

In vitro cell and tissue models for studying host-microbe interactions: a review.

PubMed

Bermudez-Brito, Miriam; Plaza-Díaz, Julio; Fontana, Luis; Muñoz-Quezada, Sergio; Gil, Angel

2013-01-01

Ideally, cell models should resemble the in vivo conditions; however, in most in vitro experimental models, epithelial cells are cultivated as monolayers, in which the establishment of functional epithelial features is not achieved. To overcome this problem, co-culture experiments with probiotics, dendritic cells and intestinal epithelial cells and three-dimensional models attempt to reconcile the complex and dynamic interactions that exist in vivo between the intestinal epithelium and bacteria on the luminal side and between the epithelium and the underlying immune system on the basolateral side. Additional models include tissue explants, bioreactors and organoids. The present review details the in vitro models used to study host-microbe interactions and explores the new tools that may help in understanding the molecular mechanisms of these interactions.

The in vitro MIMIC® platform reflects age-associated changes in immunological responses after influenza vaccination.

PubMed

Dauner, Allison; Agrawal, Pankaj; Salvatico, Jose; Tapia, Tenekua; Dhir, Vipra; Shaik, S Farzana; Drake, Donald R; Byers, Anthony M

2017-10-04

Increasing research and development costs coupled with growing concerns over healthcare expenditures necessitate the generation of pre-clinical testing models better able to predict the efficacy of vaccines, drugs and biologics. An ideal system for evaluating vaccine immunogenicity will not only be reliable but also physiologically relevant, able to be influenced by immunomodulatory characteristics such as age or previous exposure to pathogens. We have previously described a fully autologous human cell-based MIMIC® (Modular IMmune In vitro Construct) platform which enables the evaluation of innate and adaptive immunity in vitro, including naïve and recall responses. Here, we establish the ability of this module to display reduced antibody production and T cell activation upon in vitro influenza vaccination of cells from elderly adults. In the MIMIC® system, we observe a 2.7-4.2-fold reduction in strain-specific IgG production to seasonal trivalent influenza vaccine (TIV) in the elderly when compared to adults, as well as an age-dependent decline in the generation of functional antibodies. A parallel decline in IgG production with increasing age was detected via short-term ex vivo stimulation of B cells after in vivo TIV vaccination in the same cohort. Using MIMIC®, we also detect a reduction in the number but not proportion of TIV-specific multifunctional CD154 + IFNγ + IL-2 + TNFα + CD4 + T cells in elderly adults. Inefficient induction of multifunctional helper T cells with TIV stimulation in MIMIC® despite a normalized number of initial CD4 + T cells suggests a possible mechanism for an impaired anti-TIV IgG response in elderly adults. The ability of the MIMIC® system to recapitulate differential age-associated responses in vitro provides a dynamic platform for the testing of vaccine candidates and vaccine enhancement strategies in a fully human model including the ability to interrogate specific populations, such as elderly adults. Copyright © 2017 Elsevier Ltd. All rights reserved.

Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

PubMed

Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

2017-01-01

Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

Dynamic visualization the whole process of cytotoxic T lymphocytes killing the B16 tumor cells in vitro

NASA Astrophysics Data System (ADS)

Qi, Shuhong; Zhang, Zhihong

2016-03-01

Cytotoxic T lymphocytes (CTLs) played a key role in the immune system to destroy the tumor cells. Although some mechanisms of CTLs killing the tumor cells are revealed already, the dynamic information of CTLs interaction with tumor cells are still not known very clearly. Here we used confocal microscopy to visualize the whole process of CTLs killing the tumor cells in vitro. The imaging data showed that CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or some tumor cells and the average time for CTLs destroying one tumor cell is just a few minutes in vitro. The study displayed the temporal events of CTLs interacting with tumor cells at the beginning and finally killing them and directly presented the efficient tumor cell cytotoxicity of the CTLs. The results helped us to deeply understand the mechanism of the CTLs destroying the tumor cells and to develop the cancer immunotherapy.

Perspective on translating biomaterials into glioma therapy: Lessons from in vitro models

NASA Astrophysics Data System (ADS)

Cornelison, R. Chase; Munson, Jennifer M.

2018-05-01

Glioblastoma (GBM) is the most common and malignant form of brain cancer. Even with aggressive standard of care, GBM almost always recurs because its diffuse, infiltrative nature makes these tumors difficult to treat. The use of biomaterials is one strategy that has been, and is being, employed to study and overcome recurrence. Biomaterials have been used in GBM in two ways: in vitro as mediums in which to model the tumor microenvironment, and in vivo to sustain release of cytotoxic therapeutics. In vitro systems are a useful platform for studying the effects of drugs and tissue-level effectors on tumor cells in a physiologically relevant context. These systems have aided examination of how glioma cells respond to a variety of natural, synthetic, and semi-synthetic biomaterials with varying substrate properties, biochemical factor presentations, and non-malignant parenchymal cell compositions in both 2D and 3D environments. The current in vivo paradigm is completely different, however. Polymeric implants are simply used to line the post-surgical resection cavities and deliver secondary therapies, offering moderate impacts on survival. Instead, perhaps we can use the data generated from in vitro systems to design novel biomaterial-based treatments for GBM akin to a tissue engineering approach. Here we offer our perspective on the topic, summarizing how biomaterials have been used to identify facets of glioma biology in vitro and discussing the elements that show promise for translating these systems in vivo as new therapies for GBM.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo.

PubMed

Venalis, Paulius; Maurer, Britta; Akhmetshina, Alfiya; Busch, Nicole; Dees, Clara; Stürzl, Michael; Zwerina, Jochen; Jüngel, Astrid; Gay, Steffen; Schett, Georg; Distler, Oliver; Distler, Jörg H W

2009-10-01

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo

PubMed Central

Venalis, Paulius; Maurer, Britta; Akhmetshina, Alfiya; Busch, Nicole; Dees, Clara; Stürzl, Michael; Zwerina, Jochen; Jüngel, Astrid; Gay, Steffen; Schett, Georg; Distler, Oliver; Distler, Jörg HW

2009-01-01

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc. PMID:18774958

In vitro synthesis and processing of herpes simplex virus type 2 gG-2, using cell-free transcription and translation systems.

PubMed Central

Weldon, S K; Su, H K; Fetherston, J D; Courtney, R J

1990-01-01

Translation of in vitro-synthesized herpes simplex virus type 2 (HSV-2) gG-2 mRNA in a reticulocyte lysate system was used to study the processing of HSV-2 gG-2. In the presence of canine pancreatic microsomal membranes, a single species that is protected from trypsin digestion was detected. This product comigrates with the 104,000-Mr (104K) high mannose intermediate seen in HSV-2-infected-cell lysates. Endo-beta-N-acetylglucosaminidase H treatment of the in vitro-synthesized 104K protein yielded a single product migrating at 100 K. The 72K and 31K cleavage products of gG-2 were not observed in the in vitro system. These data show that the molecular weight of the nonglycosylated form of the gG-2 protein is 100,000 and that the cotranslational processing of this protein in the endoplasmic reticulum yields the 104K high-mannose intermediate. Images PMID:2154614

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

PubMed

Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

2011-12-01

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

Testing ocular irritancy in vitro with the silicon microphysiometer.

PubMed

Bruner, L H; Miller, K R; Owicki, J C; Parce, J W; Muir, V C

1991-01-01

The silicon microphysiometer, an instrument based on the light-addressable potentiometric sensor, was evaluated as an in vitro alternative for assessing ocular irritancy potential. It indirectly and non-invasively measures cell metabolism by determining the rate of acid metabolite production from cells, in this case human epidermal keratinocytes, placed inside the microphysiometer chamber. The 17 materials used for the evaluation included bar soaps, a liquid hand soap, shampoos, dishwashing liquids, laundry detergents, a fabric softener and several single chemicals. All materials tested were in liquid form. The in vivo irritancy potential of the materials was obtained from historical data using the rabbit low-volume eye test. There was a positive correlation between the in vivo irritancy potential of the test materials and the concentration of test material that decreased the acidification rate of cells by 50% (MRD(50); r = 0.86, P < 0.0001). Preliminary studies suggest other endpoints obtainable from the system may also provide useful information for making ocular safety assessments. Because the method is non-invasive, it is possible to determine whether cells recover from a treatment with the test material. The metabolic rate of the cells also increases at sub-inhibitory concentrations of some of the test materials. Because of the good correlation between the in vivo and in vitro data, the ease with which test materials can be applied to the system, and the multiple endpoints available from the system, it holds great potential as a useful in vitro alternative for ocular safety testing.

Study of Parameters Affecting the Level of Ultrasound Exposure with In Vitro Set-Ups

NASA Astrophysics Data System (ADS)

Leskinen, Jarkko J.; Hynynen, Kullervo

2010-03-01

Ultrasound (US) exposures are widely used with in vitro cell systems e.g. in stem cell and tissue engineering research. However, without the knowledge of factors affecting the level of US exposure, the outcome of the biological result may vary from test to test or even be misinterpreted. Thereby, some of the factors affecting in vitro US exposures were studied. The level of US exposure was characterized in standard commercial cell culturing plates. The temperature distributions were measured inside the wells using infrared camera and fine wire thermocouples, and pressure and intensity distributions using a laser vibrometer and a schlieren system. The measurements were made at operating frequency of around 1 MHz with varying temporal parameters and powers (up to 2 W of acoustic power). Heat accumulation between the wells varied up to 40-50% depending on the location of the well on the plate. This well-to-well variation was be linked to the activity of reporter plasmid on osteoblastic cells. Similar temperature variations within the wells were also measured. Small sub-wavelength change in the exposure distance or, respectively, liquid volume inside the well was found to alter the acoustic field in both magnitude and shape due the standing waves. The gathered data reveals the complexity of the acoustic field in a typical in vitro set-up and gives new information about the environment of the in vitro cells during US exposures. This data may be especially useful when US set-ups are designed or characterized.

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Three-dimensional in vitro cancer spheroid models for Photodynamic Therapy: Strengths and Opportunities

NASA Astrophysics Data System (ADS)

Evans, Conor

2015-03-01

Three dimensional, in vitro spheroid cultures offer considerable utility for the development and testing of anticancer photodynamic therapy regimens. More complex than monolayer cultures, three-dimensional spheroid systems replicate many of the important cell-cell and cell-matrix interactions that modulate treatment response in vivo. Simple enough to be grown by the thousands and small enough to be optically interrogated, spheroid cultures lend themselves to high-content and high-throughput imaging approaches. These advantages have enabled studies investigating photosensitizer uptake, spatiotemporal patterns of therapeutic response, alterations in oxygen diffusion and consumption during therapy, and the exploration of mechanisms that underlie therapeutic synergy. The use of quantitative imaging methods, in particular, has accelerated the pace of three-dimensional in vitro photodynamic therapy studies, enabling the rapid compilation of multiple treatment response parameters in a single experiment. Improvements in model cultures, the creation of new molecular probes of cell state and function, and innovations in imaging toolkits will be important for the advancement of spheroid culture systems for future photodynamic therapy studies.

Cell cultures in uterine leiomyomas: rapid disappearance of cells carrying MED12 mutations.

PubMed

Nadine Markowski, Dominique; Tadayyon, Mahboobeh; Bartnitzke, Sabine; Belge, Gazanfer; Maria Helmke, Burkhard; Bullerdiek, Jörn

2014-04-01

Uterine leiomyomas (UL) are the most frequent symptomatic human tumors. Nevertheless, their molecular pathogenesis is not yet fully understood. To learn more about the biology of these common neoplasms and their response to treatment, cell cultures derived from UL are a frequently used model system, but until recently appropriate genetic markers confirming their origin from the tumor cell population were lacking for most UL, i.e., those not displaying karyotypic abnormalities. The identification of MED12 mutations in the majority of UL makes it possible to trace the tumor cell population during in vitro passaging in the absence of cytogenetic abnormalities. The present study is addressing the in vitro survival of cells carrying MED12 mutations and its association with karyotypic alterations. The results challenge numerous in vitro studies into the biology and behavior of leiomyomas. Cells of one genetic subtype of UL, i.e., those with rearrangements of the high mobility AT-hook 2 protein gene (HMGA2), seem to be able to proliferate in vitro for many passages whereas tumor cells from the much more frequent MED12-mutated lesions barely survive even the first passages. Apparently, for the most frequent type of human UL no good in vitro model seems to exist because cells do not survive culturing. On the other hand, this inability may point to an Achilles' heel of this type of UL. Copyright © 2014 Wiley Periodicals, Inc.

Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues.

PubMed

Lundquist, P; Artursson, P

2016-11-15

In this contribution, we review the molecular and physiological barriers to oral delivery of peptides and nanoparticles. We discuss the opportunities and predictivity of various in vitro systems with special emphasis on human intestine in Ussing chambers. First, the molecular constraints to peptide absorption are discussed. Then the physiological barriers to peptide delivery are examined. These include the gastric and intestinal environment, the mucus barrier, tight junctions between epithelial cells, the enterocytes of the intestinal epithelium, and the subepithelial tissue. Recent data from human proteome studies are used to provide information about the protein expression profiles of the different physiological barriers to peptide and nanoparticle absorption. Strategies that have been employed to increase peptide absorption across each of the barriers are discussed. Special consideration is given to attempts at utilizing endogenous transcytotic pathways. To reliably translate in vitro data on peptide or nanoparticle permeability to the in vivo situation in a human subject, the in vitro experimental system needs to realistically capture the central aspects of the mentioned barriers. Therefore, characteristics of common in vitro cell culture systems are discussed and compared to those of human intestinal tissues. Attempts to use the cell and tissue models for in vitro-in vivo extrapolation are reviewed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Progress in Assessing Air Pollutant Risks from In Vitro Exposures: Matching Ozone Dose and Effect in Human Airway Cells

PubMed Central

Hatch, Gary E.; Duncan, Kelly E.; Diaz-Sanchez, David; Schmitt, Michael T.; Ghio, Andrew J.; Carraway, Martha Sue; McKee, John; Dailey, Lisa A.; Berntsen, Jon; Devlin, Robert B.

2014-01-01

In vitro exposures to air pollutants could, in theory, facilitate a rapid and detailed assessment of molecular mechanisms of toxicity. However, it is difficult to ensure that the dose of a gaseous pollutant to cells in tissue culture is similar to that of the same cells during in vivo exposure of a living person. The goal of the present study was to compare the dose and effect of O3 in airway cells of humans exposed in vivo to that of human cells exposed in vitro. Ten subjects breathed labeled O3 (18O3, 0.3 ppm, 2 h) while exercising intermittently. Bronchial brush biopsies and lung lavage fluids were collected 1 h post exposure for in vivo data whereas in vitro data were obtained from primary cultures of human bronchial epithelial cells exposed to 0.25–1.0 ppm 18O3 for 2 h. The O3 dose to the cells was defined as the level of 18O incorporation and the O3 effect as the fold increase in expression of inflammatory marker genes (IL-8 and COX-2). Dose and effect in cells removed from in vivo exposed subjects were lower than in cells exposed to the same 18O3 concentration in vitro suggesting upper airway O3 scrubbing in vivo. Cells collected by lavage as well as previous studies in monkeys show that cells deeper in the lung receive a higher O3 dose than cells in the bronchus. We conclude that the methods used herein show promise for replicating and comparing the in vivo dose and effect of O3 in an in vitro system. PMID:24928893

Potentiometric Biosensor for Studying Hydroquinone Cytotoxicity in vitro

PubMed Central

Wang, Yanyan; Chen, Qiang; Zeng, Xiangqun

2009-01-01

Many processes in living cells have electrochemical characteristics that are suitable for measurement by potentiometric biosensors. Potentiometric biosensors allow non invasive, real-time monitoring of the extracellular environment changes by measuring the potential at cell/sensor interface. This can be used as an indicator for overall cell cytotoxicity. The present work employs a potentiometric sensor array to investigate the cytotoxicity of hydroquinone to cultured mammalian V79 cells. Various electrode substrates (Au, PPy-HQ and PPy-PS) used for cell growth were designed and characterized. The controllable release of hydroquinone from PPy substrates was studied. Our results showed that hydroquinone exposure affected cell proliferation and delayed cell growth and attachment in a dose-dependent manner. Additionally, we have shown that exposure of V79 cells to hydroquinone at low doses (i.e 5μM) for more than 15 hours allows V79 cells to gain enhanced adaptability to survive exposure to high toxic HQ doses afterwards. Compared with traditional methods, the potentiometric biosensor not only provides non-invasive and real time monitoring of the cellular reactions but also is more sensitive for in vitro cytotoxicity study. By real time and non-invasive monitoring of the extracellular potential in vitro, the potentiometric sensor system represents a promising biosensor system for drug discovery. PMID:19926470

Biohybrid Membrane Systems and Bioreactors as Tools for In Vitro Drug Testing.

PubMed

Salerno, Simona; Bartolo, Loredana De

2017-01-01

In drug development, in vitro human model systems are absolutely essential prior to the clinical trials, considering the increasing number of chemical compounds in need of testing, and, keeping in mind that animals cannot predict all the adverse human health effects and reactions, due to the species-specific differences in metabolic pathways. The liver plays a central role in the clearance and biotransformation of chemicals and xenobiotics. In vitro liver model systems by using highly differentiated human cells could have a great impact in preclinical trials. Membrane biohybrid systems constituted of human hepatocytes and micro- and nano-structured membranes, represent valuable tools for studying drug metabolism and toxicity. Membranes act as an extracellular matrix for the adhesion of hepatocytes, and compartmentalise them in a well-defined physical and chemical microenvironment with high selectivity. Advanced 3-D tissue cultures are furthermore achieved by using membrane bioreactors (MBR), which ensure the continuous perfusion of cells protecting them from shear stress. MBRs with different configurations allow the culturing of cells at high density and under closely monitored high perfusion, similarly to the natural liver. These devices that promote the long-term maintenance and differentiation of primary human hepatocytes with preserved liver specific functions can be employed in drug testing for prolonged exposure to chemical compounds and for assessing repeated-dose toxicity. The use of primary human hepatocytes in MBRs is the only system providing a faster and more cost-effective method of analysis for the prediction of in vitro human drug metabolism and enzyme induction alternative and/or complementary to the animal experimentation. In this paper, in vitro models for studying drug metabolism and toxicity as advanced biohybrid membrane systems and MBRs will be reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.

PubMed

Cottet, Sandra; Corthésy-Theulaz, Irène; Spertini, François; Corthésy, Blaise

2002-09-13

Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.

Three-dimensional hydrogel scaffolds facilitate in vitro self-renewal of human skin-derived precursors.

PubMed

Wang, Xinyue; Liu, Shu; Zhao, Qian; Li, Na; Zhang, Huishan; Zhang, Xudong; Lei, Xiaohua; Zhao, Huashan; Deng, Zhili; Qiao, Jingqiao; Cao, Yujing; Ning, Lina; Liu, Shuang; Duan, Enkui

2014-07-01

Skin-derived precursors (SKPs) are multipotent cells with dermal stem cell properties. These easily available cells possess the capacity to reconstitute the skin in vivo, as well as a broader differentiation potential in vitro, which endows them with great prospects in regenerative medicine. However, the present authors' group and others previously found that adult human SKPs (hSKPs) expanded deficiently in vitro, which largely counteracted their research and practical values. Taking the physiological micro-environment of hSKPs into consideration, the authors sought to establish a hydrogel scaffold-based three-dimensional (3-D) culture system for hSKPs in the present study. After comparing their morphology, growth characteristics, signature gene expression and differentiation potential in different hydrogels, the present authors found that a chemically defined hyaluronic acid and denatured collagen-based hydrogel system that mimicked the natural niche of hSKPs in the dermis could alleviate hSKP senescence, support hSKP proliferation as spheres, while largely retaining their properties and potential. This study suggested that recapitulating the in vivo stem cell niche by providing them with 3-D extracellular matrix environments could help them achieve better self-renewal in vitro. In addition, the animal-origin-free and biocompatible 3-D hydrogel system will certainly benefit fundamental research and clinical applications of hSKPs in the near future. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Silibinin and indocyanine green-loaded nanoparticles inhibit the growth and metastasis of mammalian breast cancer cells in vitro.

PubMed

Sun, Hui-Ping; Su, Jing-Han; Meng, Qing-Shuo; Yin, Qi; Zhang, Zhi-Wen; Yu, Hai-Jun; Zhang, Peng-Cheng; Wang, Si-Ling; Li, Ya-Ping

2016-07-01

To improve the therapeutic efficacy of cancer treatments, combinational therapies based on nanosized drug delivery system (NDDS) has been developed recently. In this study we designed a new NDDS loaded with an anti-metastatic drug silibinin and a photothermal agent indocyanine green (ICG), and investigated its effects on the growth and metastasis of breast cancer cells in vitro. Silibinin and ICG were self-assembled into PCL lipid nanoparticles (SIPNs). Their physical characteristics including the particle size, zeta potential, morphology and in vitro drug release were examined. 4T1 mammalian breast cancer cells were used to evaluate their cellular internalization, cytotoxicity, and their influences on wound healing, in vitro cell migration and invasion. SIPNs showed a well-defined spherical shape with averaged size of 126.3±0.4 nm and zeta potential of -10.3±0.2 mV. NIR laser irradiation substantially increased the in vitro release of silibinin from the SIPNs (58.3% at the first 8 h, and 97.8% for the total release). Furthermore, NIR laser irradiation markedly increased the uptake of SIPNs into 4T1 cells. Under the NIR laser irradiation, both SIPNs and IPNs (PCL lipid nanoparticles loaded with ICG alone) caused dose-dependent ablation of 4T1 cells. The wound healing, migration and invasion experiments showed that SIPNs exposed to NIR laser irradiation exhibited dramatic in vitro anti-metastasis effects. SIPNs show temperature-sensitive drug release following NIR laser irradiation, which can inhibit the growth and metastasis of breast cancer cells in vitro.

Translating neurobehavioural endpoints of developmental neurotoxicity tests into in vitro assays and readouts

PubMed Central

van Thriel, Christoph; Westerink, Remco; Beste, Christian; Bale, Ambuja S.; Lein, Pamela J.; Leist, Marcel

2011-01-01

The developing nervous system is particularly vulnerable to chemical insults. Exposure to chemicals can results in neurobehavioural alterations, and these have been be used as sensitive readouts to assess neurotoxicity in animals and man. Deconstructing neurobehaviour into relevant cellular and molecular components may allow for detection of specific neurotoxic effects in cell-based systems, which in turn may allow an easier examination of neurotoxic pathways and modes of actions and eventually inform the regulatory assessment of chemicals with potential developmental neurotoxicity. Here, current developments towards these goals are reviewed. Imaging genetics (CB) provides new insights into the neurobiological correlates of cognitive function that are being used to delineate neurotoxic mechanisms. The gaps between in vivo neurobehaviour and real-time in vitro measurements of neuronal function are being bridged by ex vivo measurements of synaptic plasticity (RW). An example of solvent neurotoxicity demonstrates how an in vivo neurological defect can be linked via the N-methyl-D-aspartate (NMDA)-glutamate receptor as a common target to in vitro readouts (AB). Axonal and dendritic morphology in vitro proved to be good correlates of neuronal connectivity and neurobehaviour in animals exposed to polychlorinated biphenyls and organophosphorus pesticides (PJL). Similarly, chemically-induced changes in neuronal morphology affected the formation of neuronal networks on structured surfaces. Such network formation may become an important readout for developmental neurotoxicity in vitro (CvT), especially when combined with human neurons derived from embryonic stem cells (ML). We envision that future in vitro test systems for developmental neurotoxicity will combine the above approaches with exposure information, and we suggest a strategy for test system development and cell-based risk assessment. PMID:22008243

Long-term microfluidic glucose and lactate monitoring in hepatic cell culture

PubMed Central

Prill, Sebastian; Jaeger, Magnus S.; Duschl, Claus

2014-01-01

Monitoring cellular bioenergetic pathways provides the basis for a detailed understanding of the physiological state of a cell culture. Therefore, it is widely used as a tool amongst others in the field of in vitro toxicology. The resulting metabolic information allows for performing in vitro toxicology assays for assessing drug-induced toxicity. In this study, we demonstrate the value of a microsystem for the fully automated detection of drug-induced changes in cellular viability by continuous monitoring of the metabolic activity over several days. To this end, glucose consumption and lactate secretion of a hepatic tumor cell line were continuously measured using microfluidically addressed electrochemical sensors. Adapting enzyme-based electrochemical flat-plate sensors, originally designed for human whole-blood samples, to their use with cell culture medium supersedes the common manual and laborious colorimetric assays and off-line operated external measurement systems. The cells were exposed to different concentrations of the mitochondrial inhibitor rotenone and the cellular response was analyzed by detecting changes in the rates of the glucose and lactate metabolism. Thus, the system provides real-time information on drug-induced liver injury in vitro. PMID:24926387

Controlled release of paclitaxel from a self-assembling peptide hydrogel formed in situ and antitumor study in vitro

PubMed Central

Liu, Jingping; Zhang, Lanlan; Yang, Zehong; Zhao, Xiaojun

2011-01-01

Background A nanoscale injectable in situ-forming hydrogel drug delivery system was developed in this study. The system was based on a self-assembling peptide RADA16 solution, which can spontaneously form a hydrogel rapidly under physiological conditions. We used the RADA16 hydrogel for the controlled release of paclitaxel (PTX), a hydrophobic antitumor drug. Methods The RADA16-PTX suspension was prepared simply by magnetic stirring, followed by atomic force microscopy, circular dichroism analysis, dynamic light scattering, rheological analysis, an in vitro release assay, and a cell viability test. Results The results indicated that RADA16 and PTX can interact with each other and that the amphiphilic peptide was able to stabilize hydrophobic drugs in aqueous solution. The particle size of PTX was markedly decreased in the RADA16 solution compared with its size in water. The RADA16-PTX suspension could form a hydrogel in culture medium, and the elasticity of the hydrogel showed a positive correlation with peptide concentration. In vitro release measurements indicated that hydrogels with a higher peptide concentration had a longer half-release time. The RADA16-PTX hydrogel could effectively inhibit the growth of the breast cancer cell line, MDA-MB-435S, in vitro, and hydrogels with higher peptide concentrations were more effective at inhibiting tumor cell proliferation. The RADA16-PTX hydrogel was effective at controlling the release of PTX and inhibiting tumor cell growth in vitro. Conclusion Self-assembling peptide hydrogels may work well as a system for drug delivery. PMID:22114478

Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

PubMed Central

Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

2015-01-01

Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

Specific immunotherapy of experimental myasthenia gravis in vitro and in vivo: the Guided Missile strategy.

PubMed

Sun, W; Adams, R N; Miagkov, A; Lu, Y; Juon, H-S; Drachman, D B

2012-10-15

Current immunotherapy of myasthenia gravis (MG) is often effective, but entails risks of infection and neoplasia. The "Guided Missile" strategy described here is designed to target and eliminate the individual's unique AChR-specific T cell repertoire, without otherwise interfering with the immune system. We genetically engineered dendritic cells to present AChR epitopes and simultaneously express Fas ligand in an ongoing EAMG model. In both in vitro and in vivo experiments, these engineered cells specifically killed AChR-responsive T cells without otherwise damaging the immune system. AChR antibodies were markedly reduced in the treated mice. Translation of this method to treat human MG is possible. Copyright © 2012 Elsevier B.V. All rights reserved.

Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

PubMed

Galea, Gabriel L; Price, Joanna S

2015-01-01

Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus.

PubMed

Lew, Y Y; Michalak, T I

2001-02-01

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.

Effect of Citrus bergamia juice on human neuroblastoma cells in vitro and in metastatic xenograft models.

PubMed

Navarra, M; Ursino, M R; Ferlazzo, N; Russo, M; Schumacher, U; Valentiner, U

2014-06-01

Neuroblastoma is the most common extracranial pediatric solid tumor with poor prognosis in children with disseminated stage of disease. A number of studies show that molecules largely distributed in commonly consumed fruits and vegetables may have anti-tumor activity. In this study we evaluate the effect of Citrus bergamia (bergamot) juice (BJ) in vitro and in a spontaneous metastatic neuroblastoma SCID mouse model. Qualitative and quantitative characterizations of BJ flavonoid fractions were performed by RP-HPLC/PDA/MS. We show that BJ significantly affects SK-N-SH and LAN-1 cell proliferation in vitro, but fails to reduce primary tumor weight in vivo. Moreover, BJ reduced cell adhesiveness and invasion of LAN-1 and SK-N-SH cells in vitro and the number of pulmonary metastases under consideration of the number of tumor cells in the blood in mice inoculated with LAN-1 cells in vivo. These effects without any apparent sign of systemic toxicity confirm the potential clinical interest of BJ and lay the basis for further investigation in cancer. Copyright © 2014. Published by Elsevier B.V.

Gene Transfer by Guanidinium-Cholesterol Cationic Lipids into Airway Epithelial Cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Oudrhiri, Noufissa; Vigneron, Jean-Pierre; Peuchmaur, Michel; Leclerc, Tony; Lehn, Jean-Marie; Lehn, Pierre

1997-03-01

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis (guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes.

PubMed

Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan M W

2006-05-25

Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable.

In vitro osteogenesis of human stem cells by using a three-dimensional perfusion bioreactor culture system: a review.

PubMed

Ceccarelli, Gabriele; Bloise, Nora; Vercellino, Marco; Battaglia, Rosalia; Morgante, Lucia; De Angelis, Maria Gabriella Cusella; Imbriani, Marcello; Visai, Livia

2013-04-01

Tissue engineering (by culturing cells on appropriate scaffolds, and using bioreactors to drive the correct bone structure formation) is an attractive alternative to bone grafting or implantation of bone substitutes. Osteogenesis is a biological process that involves many molecular intracellular pathways organized to optimize bone modeling. The use of bioreactor systems and especially the perfusion bioreactor, provides both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In this mini-review all the characteristics for the production of an appropriate bone construct are analyzed: the stem cell source, scaffolds useful for the seeding of pre-osteoblastic cells and the effects of fluid flow on differentiation and proliferation of bone precursor cells. By automating and standardizing tissue manufacture in controlled closed systems, engineered tissues may reduce the gap between the process of bone formation in vitro and subsequent graft of bone substitutes in vivo.

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo

PubMed Central

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M.; Brennan, Patrick J.; Banerjee, Pinaki P.; Wiener, Susan J.; Orange, Jordan S.; Brenner, Michael B.; Grupp, Stephan A.; Nichols, Kim E.

2013-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we find that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially-induced by iNKT cell agonists of varying TCR affinities, such as OCH, α-galactosyl ceramide and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of T cell receptor (TCR) signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell-deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T-lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T-lymphoma. PMID:24563871

Ascorbyl palmitate/d-α-tocopheryl polyethylene glycol 1000 succinate monoester mixed micelles for prolonged circulation and targeted delivery of compound K for antilung cancer therapy in vitro and in vivo

PubMed Central

Zhang, Youwen; Tong, Deyin; Che, Daobiao; Pei, Bing; Xia, Xiaodong; Yuan, Gaofeng; Jin, Xin

2017-01-01

The roles of ginsenoside compound K (CK) in inhibiting tumor have been widely recognized in recent years. However, low water solubility and significant P-gp efflux have restricted its application. In this study, CK ascorbyl palmitate (AP)/d-α-tocopheryl polyethylene glycol 1000 succinate monoester (TPGS) mixed micelles were prepared as a delivery system to increase the absorption and targeted antitumor effect of CK. Consequently, the solubility of CK increased from 35.2±4.3 to 1,463.2±153.3 μg/mL. Furthermore, in an in vitro A549 cell model, CK AP/TPGS mixed micelles significantly inhibited cell growth, induced G0/G1 phase cell cycle arrest, induced cell apoptosis, and inhibited cell migration compared to free CK, all indicating that the developed micellar delivery system could increase the antitumor effect of CK in vitro. Both in vitro cellular fluorescence uptake and in vivo near-infrared imaging studies indicated that AP/TPGS mixed micelles can promote cellular uptake and enhance tumor targeting. Moreover, studies in the A549 lung cancer xenograft mouse model showed that CK AP/TPGS mixed micelles are an efficient tumor-targeted drug delivery system with an effective antitumor effect. Western blot analysis further confirmed that the marked antitumor effect in vivo could likely be due to apoptosis promotion and P-gp efflux inhibition. Therefore, these findings suggest that the AP/TPGS mixed micellar delivery system could be an efficient delivery strategy for enhanced tumor targeting and antitumor effects. PMID:28144142

Neonatal rat heart cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

1997-01-01

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

Novel in vitro and mathematical models for the prediction of chemical toxicity.

PubMed

Williams, Dominic P; Shipley, Rebecca; Ellis, Marianne J; Webb, Steve; Ward, John; Gardner, Iain; Creton, Stuart

2013-01-01

The focus of much scientific and medical research is directed towards understanding the disease process and defining therapeutic intervention strategies. The scientific basis of drug safety is very complex and currently remains poorly understood, despite the fact that adverse drug reactions (ADRs) are a major health concern and a serious impediment to development of new medicines. Toxicity issues account for ∼21% drug attrition during drug development and safety testing strategies require considerable animal use. Mechanistic relationships between drug plasma levels and molecular/cellular events that culminate in whole organ toxicity underpins development of novel safety assessment strategies. Current in vitro test systems are poorly predictive of toxicity of chemicals entering the systemic circulation, particularly to the liver. Such systems fall short because of (1) the physiological gap between cells currently used and human hepatocytes existing in their native state, (2) the lack of physiological integration with other cells/systems within organs, required to amplify the initial toxicological lesion into overt toxicity, (3) the inability to assess how low level cell damage induced by chemicals may develop into overt organ toxicity in a minority of patients, (4) lack of consideration of systemic effects. Reproduction of centrilobular and periportal hepatocyte phenotypes in in vitro culture is crucial for sensitive detection of cellular stress. Hepatocyte metabolism/phenotype is dependent on cell position along the liver lobule, with corresponding differences in exposure to substrate, oxygen and hormone gradients. Application of bioartificial liver (BAL) technology can encompass in vitro predictive toxicity testing with enhanced sensitivity and improved mechanistic understanding. Combining this technology with mechanistic mathematical models describing intracellular metabolism, fluid-flow, substrate, hormone and nutrient distribution provides the opportunity to design the BAL specifically to mimic the in vivo scenario. Such mathematical models enable theoretical hypothesis testing, will inform the design of in vitro experiments, and will enable both refinement and reduction of in vivo animal trials. In this way, development of novel mathematical modelling tools will help to focus and direct in vitro and in vivo research, and can be used as a framework for other areas of drug safety science.

Novel in vitro and mathematical models for the prediction of chemical toxicity

PubMed Central

Shipley, Rebecca; Ellis, Marianne J.; Webb, Steve; Ward, John; Gardner, Iain; Creton, Stuart

2013-01-01

The focus of much scientific and medical research is directed towards understanding the disease process and defining therapeutic intervention strategies. The scientific basis of drug safety is very complex and currently remains poorly understood, despite the fact that adverse drug reactions (ADRs) are a major health concern and a serious impediment to development of new medicines. Toxicity issues account for ∼21% drug attrition during drug development and safety testing strategies require considerable animal use. Mechanistic relationships between drug plasma levels and molecular/cellular events that culminate in whole organ toxicity underpins development of novel safety assessment strategies. Current in vitro test systems are poorly predictive of toxicity of chemicals entering the systemic circulation, particularly to the liver. Such systems fall short because of (1) the physiological gap between cells currently used and human hepatocytes existing in their native state, (2) the lack of physiological integration with other cells/systems within organs, required to amplify the initial toxicological lesion into overt toxicity, (3) the inability to assess how low level cell damage induced by chemicals may develop into overt organ toxicity in a minority of patients, (4) lack of consideration of systemic effects. Reproduction of centrilobular and periportal hepatocyte phenotypes in in vitro culture is crucial for sensitive detection of cellular stress. Hepatocyte metabolism/phenotype is dependent on cell position along the liver lobule, with corresponding differences in exposure to substrate, oxygen and hormone gradients. Application of bioartificial liver (BAL) technology can encompass in vitro predictive toxicity testing with enhanced sensitivity and improved mechanistic understanding. Combining this technology with mechanistic mathematical models describing intracellular metabolism, fluid-flow, substrate, hormone and nutrient distribution provides the opportunity to design the BAL specifically to mimic the in vivo scenario. Such mathematical models enable theoretical hypothesis testing, will inform the design of in vitro experiments, and will enable both refinement and reduction of in vivo animal trials. In this way, development of novel mathematical modelling tools will help to focus and direct in vitro and in vivo research, and can be used as a framework for other areas of drug safety science. PMID:26966512

A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

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

Webb, Carol F., E-mail: carol-webb@omrf.org; Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK; Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights:more » • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.« less

Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

PubMed

Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

2017-01-01

Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo .

PubMed

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M; Brennan, Patrick J; Banerjee, Pinaki P; Wiener, Susan J; Orange, Jordan S; Brenner, Michael B; Grupp, Stephan A; Nichols, Kim E

2014-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. ©2013 AACR.

[Establishment and application of mechanical strain loading system of multi-channel cells].

PubMed

Li, Yongming; Wang, Hua; Zhang, Xiaodong; Tang, Lin

2012-02-01

Based on single-chip microcomputer, we have established a mechanical strain loading system with multi-channel to study the biological behavior of cultured cells in vitro under mechanical strain. We developed a multi-channel cell strain loading device controlled by single-chip microcomputer. We controlled the vacuum pump with vacuum chamber to make negative pressure changing periodically in the vacuum chamber. The tested cells were seeded on the surface of an elastic membrane mounted on the vacuum chamber, and could be strained or relaxed by cyclic pressure. Since the cells are attached to the surface of the membrane, they presumably experience the same deformation as that was applied to the membrane. The system was easy to carry and to operate, with deformation rate (1%-21%) and frequency (0-0. 5Hz) which could be adjusted correctly according to experimental requirement, and could compare different deformation rate of three channels at the same time. The system ran stably and completely achieved design aims, and provided a method to study the biological behavior of cultured cells attached to the surface of the elastic membrane under mechanical strain in vitro.

In vitro transcription in E. coli crude lysates prepared on cellophane discs.

PubMed Central

Valla, S; Lindqvist, B H

1978-01-01

An in vitro RNA-synthesizing system consisting of gently lysed E. coli cells on cellophane discs is described. The system has been optimalized with respect to total RNA synthesis. Under certain standard conditions DNA dependent RNA polymerase (EC 2.7.7.6) is responsible for the majority of the RNA synthesis. The extensive rifampicin sensitivity of the synthesis indicates that most of the transcripts are initiated in vitro. The RNA synthesizing system described here has been developed with the aim of studying phage transcription in vitro. We show here that lysates of a P4 infected P2 lysogen support initiation and propagation of transcription from the P2 prophage. PMID:27767

Engineered in vitro disease models.

PubMed

Benam, Kambez H; Dauth, Stephanie; Hassell, Bryan; Herland, Anna; Jain, Abhishek; Jang, Kyung-Jin; Karalis, Katia; Kim, Hyun Jung; MacQueen, Luke; Mahmoodian, Roza; Musah, Samira; Torisawa, Yu-suke; van der Meer, Andries D; Villenave, Remi; Yadid, Moran; Parker, Kevin K; Ingber, Donald E

2015-01-01

The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.

Computer Simulation of Embryonic Systems: What can a ...

EPA Pesticide Factsheets

(1) Standard practice for assessing developmental toxicity is the observation of apical endpoints (intrauterine death, fetal growth retardation, structural malformations) in pregnant rats/rabbits following exposure during organogenesis. EPA’s computational toxicology research program (ToxCast) generated vast in vitro cellular and molecular effects data on >1858 chemicals in >600 high-throughput screening (HTS) assays. The diversity of assays has been increased for developmental toxicity with several HTS platforms, including the devTOX-quickPredict assay from Stemina Biomarker Discovery utilizing the human embryonic stem cell line (H9). Translating these HTS data into higher order-predictions of developmental toxicity is a significant challenge. Here, we address the application of computational systems models that recapitulate the kinematics of dynamical cell signaling networks (e.g., SHH, FGF, BMP, retinoids) in a CompuCell3D.org modeling environment. Examples include angiogenesis (angiodysplasia) and dysmorphogenesis. Being numerically responsive to perturbation, these models are amenable to data integration for systems Toxicology and Adverse Outcome Pathways (AOPs). The AOP simulation outputs predict potential phenotypes based on the in vitro HTS data ToxCast. A heuristic computational intelligence framework that recapitulates the kinematics of dynamical cell signaling networks in the embryo, together with the in vitro profiling data, produce quantitative pr

In Vitro Analysis of the Role of Schwann Cells on Axonal Degeneration and Regeneration Using Sensory Neurons from Dorsal Root Ganglia.

PubMed

López-Leal, Rodrigo; Diaz, Paula; Court, Felipe A

2018-01-01

Sensory neurons from dorsal root ganglion efficiently regenerate after peripheral nerve injuries. These neurons are widely used as a model system to study degenerative mechanisms of the soma and axons, as well as regenerative axonal growth in the peripheral nervous system. This chapter describes techniques associated to the study of axonal degeneration and regeneration using explant cultures of dorsal root ganglion sensory neurons in vitro in the presence or absence of Schwann cells. Schwann cells are extremely important due to their involvement in tissue clearance during axonal degeneration as well as their known pro-regenerative effect during regeneration in the peripheral nervous system. We describe methods to induce and study axonal degeneration triggered by axotomy (mechanical separation of the axon from its soma) and treatment with vinblastine (which blocks axonal transport), which constitute clinically relevant mechanical and toxic models of axonal degeneration. In addition, we describe three different methods to evaluate axonal regeneration using quantitative methods. These protocols constitute a valuable tool to analyze in vitro mechanisms associated to axonal degeneration and regeneration of sensory neurons and the role of Schwann cells in these processes.

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

Harada, M.; Odaka, K.; Kondo, K.

The effects of activated lymphocytes were studied in the regulation of in vitro hematopoiesis. Peripheral blood lymphocytes stimulated by concanavalin A (Con A) were cocultured with normal bone marrow cells in the assay system of hematopoietic stem cells. Con-A-stimulated lymphocytes and their supernatants showed significant suppression of in vitro growth of myeloid and erythroid progenitor cells (CFU-C, CFU-E, and BFU-E). Suppressive activity detected in the T-cell fraction was completely abolished by treatment with OKT3 or OKT8 monoclonal antibody and complement and 20 Gy radiation but not OKT4 or OKIa1 antibody and complement. These observations indicate that peripheral blood lymphocytes canmore » be induced by Con-A stimulation to become suppressor T cells for myeloid and erythroid progenitor cells that are OKT8 positive, Ia negative, and radiosensitive. Together with our previous observation that CFU-C suppressor cells induced by alloantigen stimulation are radioresistant and OKT8- and Ia-positive T cells, it is suggested that in vitro hematopoiesis may be regulated by heterogeneous subpopulations of activated T-lymphocytes.« less

The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo.

PubMed

Okada, Masashi; Kuramoto, Kenta; Takeda, Hiroyuki; Watarai, Hikaru; Sakaki, Hirotsugu; Seino, Shizuka; Seino, Manabu; Suzuki, Shuhei; Kitanaka, Chifumi

2016-05-10

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted.

Aluminum–phthalocyanine chloride associated to poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as a new third-generation photosensitizer for anticancer photodynamic therapy

PubMed Central

Muehlmann, Luis Alexandre; Ma, Beatriz Chiyin; Longo, João Paulo Figueiró; Almeida Santos, Maria de Fátima Menezes; Azevedo, Ricardo Bentes

2014-01-01

Photodynamic therapy is generally considered to be safer than conventional anticancer therapies, and it is effective against different kinds of cancer. However, its clinical application has been significantly limited by the hydrophobicity of photosensitizers. In this work, a system composed of the hydrophobic photosensitizer aluminum–phthalocyanine chloride (AlPc) associated with water dispersible poly(methyl vinyl ether-co-maleic anhydride) nanoparticles is described. AlPc was associated with nanoparticles produced by a method of solvent displacement. This system was analyzed for its physicochemical characteristics, and for its photodynamic activity in vitro in cancerous (murine mammary carcinoma cell lineage 4T1, and human mammary adenocarcinoma cells MCF-7) and noncancerous (murine fibroblast cell lineage NIH/3T3, and human mammary epithelial cell lineage MCF-10A) cell lines. Cell viability and the elicited mechanisms of cell death were evaluated after the application of photodynamic therapy. This system showed improved photophysical and photochemical properties in aqueous media in comparison to the free photosensitizer, and it was effective against cancerous cells in vitro. PMID:24634582

Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

PubMed

Choi, Yoon Pyo; Lee, Joo Hyun; Gao, Ming-Qing; Kim, Baek Gil; Kang, Suki; Kim, Se Hoon; Cho, Nam Hoon

2014-11-01

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5β1 and αvβ3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis. © 2014 UICC.

[Immortalization of erythroid progenitors for in vitro large-scale red cell production].

PubMed

Caulier, A; Guyonneau Harmand, L; Garçon, L

2017-09-01

Population ageing and increase in cancer incidence may lead to a decreased availability of red blood cell units. Thus, finding an alternative source of red blood cells is a highly relevant challenge. The possibility to reproduce in vitro the human erythropoiesis opens a new era, particularly since the improvement in the culture systems allows to produce erythrocytes from induced-Pluripotent Stem Cells (iPSCs), or CD34 + Hematopoietic Stem Cells (HSCs). iPSCs have the advantage of in vitro self-renewal, but lead to poor amplification and maturation defects (high persistence of nucleated erythroid precursors). Erythroid differentiation from HSC allows a far better amplification and adult-like hemoglobin synthesis. But the inability of these progenitors to self-renew in vitro remains a limit in their use as a source of stem cells. A major improvement would consist in immortalizing these erythroid progenitors so that they could expand indefinitively. Inducible transgenesis is the first way to achieve this goal. To date, the best immortalized-cell models involve strong oncogenes induction, such as c-Myc, Bcl-xL, and mostly E6/E7 HPV16 viral oncoproteins. However, the quality of terminal differentiation of erythroid progenitors generated by these oncogenes is not optimal yet and the long-term stability of such systems is unknown. Moreover, viral transgenesis and inducible expression of oncogenes raise important problems in term of safety, since the enucleation rate is not 100% and no nucleated cells having replicative capacities should be present in the final product. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

[In vitro cell culture technology in cosmetology research].

PubMed

Gojniczek, Katarzyna; Garncarczyk, Agnieszka; Pytel, Agata

2005-01-01

For ages the humanity has been looking for all kind of active substances, which could be used in improving the health and the appearance of our skin. People try to find out how to protect the skin from harmful, environmental factors. Every year a lot of new natural and synthetic, chemical substances are discovered. All of them potentially could be used as a cosmetic ingredient. In cosmetology research most of new xenobiotics were tested in vivo on animals. Alternative methods to in vivo tests are in vitro tests with skin cell culture system. The aim of this work was to describe two-dimensional and tree-dimensional skin cell cultures. Additionally, in this work we wanted to prove the usefulness of in vitro skin cell cultures in cosmetology research.

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Nikolov, Ivan; Skutella, Thomas; Just, Lothar

2007-01-01

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

Towards the engineering of in vitro systems.

PubMed

Hold, Christoph; Panke, Sven

2009-08-06

Synthetic biology aims at rationally implementing biological systems from scratch. Given the complexity of living systems and our current lack of understanding of many aspects of living cells, this is a major undertaking. The design of in vitro systems can be considerably easier, because they can consist of fewer constituents, are quasi time invariant, their parameter space can be better accessed and they can be much more easily perturbed and then analysed chemically and mathematically. However, even for simplified in vitro systems, following a comprehensively rational design procedure is still difficult. When looking at a comparatively simple system, such as a medium-sized enzymatic reaction network as it is represented by glycolysis, major issues such as a lack of comprehensive enzyme kinetics and of suitable knowledge on crucial design parameters remain. Nevertheless, in vitro systems are very suitable to overcome these obstacles and therefore well placed to act as a stepping stone to engineering living systems.

Intracellular Trafficking of Baculovirus Particles: A Quantitative Study of the HearNPV/HzAM1 Cell and AcMNPV/Sf9 Cell Systems.

PubMed

Matindoost, Leila; Nielsen, Lars K; Reid, Steve

2015-05-05

To replace the in vivo production of baculovirus-based biopesticides with a more convenient in vitro produced product, the limitations imposed by in vitro production have to be solved. One of the main problems is the low titer of HearNPV budded virions (BV) in vitro as the use of low BV titer stocks can result in non-homogenous infections resulting in multiple virus replication cycles during scale up that leads to low Occlusion Body yields. Here we investigate the baculovirus traffic in subcellular fractions of host cells throughout infection with an emphasis on AcMNPV/Sf9 and HearNPV/HzAM1 systems distinguished as "good" and "bad" BV producers, respectively. qPCR quantification of viral DNA in the nucleus, cytoplasm and extracellular fractions demonstrated that although the HearNPV/HzAM1 system produces twice the amount of vDNA as the AcMNPV/Sf9 system, its percentage of BV to total progeny vDNA was lower. vDNA egress from the nucleus to the cytoplasm is sufficient in both systems, however, a higher percentage of vDNA in the HearNPV/HzAM1 system remain in the cytoplasm and do not bud out of the cells compared to the AcMNPV/Sf9 system. In both systems more than 75% of the vDNA produced in the nuclear fraction go unused, without budding or being encapsulated in OBs showing the capacity for improvements that could result from the engineering of the virus/cell line systems to achieve better productivities for both BV and OB yields.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

PubMed

Lee, Se Jeong; Kang, Won Young; Yoon, Yeup; Jin, Ju Youn; Song, Hye Jin; Her, Jung Hyun; Kang, Sang Mi; Hwang, Yu Kyeong; Kang, Kyeong Jin; Joo, Kyeung Min; Nam, Do-Hyun

2015-12-24

Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

In vitro reestablishment of cell-cell contacts in adult rat cardiomyocytes. Functional role of transmembrane components in the formation of new intercalated disk-like cell contacts.

PubMed

Eppenberger, H M; Zuppinger, C

1999-01-01

Primary adult rat cardiomyocytes (ARC)in culture are shown to be a model system for cardiac cell hypertrophy in vitro. ARC undergo a process of morphological transformation and grow only by increase in cell size, however, without loss of the cardiac phenotype. The isolated cells spread and establish new cell-cell contacts, eventually forming a two-dimensional heart tissue-like synchronously beating cell sheet. The reformation of specific cell contacts (intercalated disks) is shown also between ventricular and atrial cardiomyocytes by using antibodies against the gap junction protein connexin-43 and after microinjection into ARC of N-cadherin cDNA fused to reporter green fluorescent protein (GFP) cDNA. The expressed fusion protein allowed the study of live cell cultures and of the dynamics of the adherens junction protein N-cadherin during the formation of new cell-cell contacts. The possible use of the formed ARC cell-sheet cells under microgravity conditions as a test system for the reformation of the cytoskeleton of heart muscle cells is proposed.

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.

Diving under a microscope--a new simple and versatile in vitro diving device for fluorescence and confocal microscopy allowing the controls of hydrostatic pressure, gas pressures, and kinetics of gas saturation.

PubMed

Wang, Qiong; Belhomme, Marc; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Theron, Michaël

2013-06-01

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

PubMed Central

Isherwood, Beverley; Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I; Canel, Marta; Serrels, Alan; Brunton, Valerie G; Carragher, Neil O

2011-01-01

Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates. PMID:24310493

Bidirectional communication between sensory neurons and osteoblasts in an in vitro coculture system.

PubMed

Kodama, Daisuke; Hirai, Takao; Kondo, Hisataka; Hamamura, Kazunori; Togari, Akifumi

2017-02-01

Recent studies have revealed that the sensory nervous system is involved in bone metabolism. However, the mechanism of communication between neurons and osteoblasts is yet to be elucidated. In this study, we investigated the signaling pathways between sensory neurons of the dorsal root ganglion (DRG) and the osteoblast-like MC3T3-E1 cells using an in vitro coculture system. Our findings indicate that signal transduction from DRG-derived neurons to MC3T3-E1 cells is suppressed by antagonists of the AMPA receptor and the NK 1 receptor. Conversely, signal transduction from MC3T3-E1 cells to DRG-derived neurons is suppressed by a P2X 7 receptor antagonist. Our results suggest that these cells communicate with each other by exocytosis of glutamate, substance P in the efferent signal, and ATP in the afferent signal. © 2017 Federation of European Biochemical Societies.

Quantifying Osteogenic Cell Degradation of Silk Biomaterials

PubMed Central

Sengupta, Sejuti; Park, Sang-Hyug; Seok, Gil Eun; Patel, Atur; Numata, Keiji; Lu, Chia-Li; Kaplan, David L.

2010-01-01

The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins α5 and β1 while the osteoclasts upregulated integrins α2 and β1. There was significant contrast in responses on the silk matrices between osteogenic cells vs undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing and physiological state and the ability to better understand the role of different cell types is critical to overall regenerative outcomes. PMID:21105641

Neuromuscular junction formation between human stem-cell-derived motoneurons and rat skeletal muscle in a defined system.

PubMed

Guo, Xiufang; Das, Mainak; Rumsey, John; Gonzalez, Mercedes; Stancescu, Maria; Hickman, James

2010-12-01

To date, the coculture of motoneurons (MNs) and skeletal muscle in a defined in vitro system has only been described in one study and that was between rat MNs and rat skeletal muscle. No in vitro studies have demonstrated human MN to rat muscle synapse formation, although numerous studies have attempted to implant human stem cells into rat models to determine if they could be of therapeutic use in disease or spinal injury models, although with little evidence of neuromuscular junction (NMJ) formation. In this report, MNs differentiated from human spinal cord stem cells, together with rat skeletal myotubes, were used to build a coculture system to demonstrate that NMJ formation between human MNs and rat skeletal muscles is possible. The culture was characterized by morphology, immunocytochemistry, and electrophysiology, while NMJ formation was demonstrated by immunocytochemistry and videography. This defined system provides a highly controlled reproducible model for studying the formation, regulation, maintenance, and repair of NMJs. The in vitro coculture system developed here will be an important model system to study NMJ development, the physiological and functional mechanism of synaptic transmission, and NMJ- or synapse-related disorders such as amyotrophic lateral sclerosis, as well as for drug screening and therapy design.

Correlation between in vivo and in vitro pulmonary responses to jet propulsion fuel-8 using precision-cut lung slices and a dynamic organ culture system.

PubMed

Hays, Allison M; Lantz, R Clark; Witten, Mark L

2003-01-01

In tissue slice models, interactions between the heterogeneous cell types comprising the lung parenchyma are maintained thus providing a controlled system for the study of pulmonary toxicology in vitro. However, validation of the model in vitro system must be affirmed. Previous reports, in in vivo systems, have demonstrated that Clara cells and alveolar type II cells are the targets following inhalation of JP-8 jet fuel. We have utilized the lung slice model to determine if cellular targets are similar following in vitro exposure to JP-8. Agar-filled adult rat lung explants were cored and precision cut, using the Brende/Vitron tissue slicer. Slices were cultured on titanium screens located as half-cylinders in cylindrical Teflon cradles that were loaded into standard scintillation vials and incubated at 37 degrees C. Slices were exposed to JP-8 jet fuel (0.5 mg/ml, 1.0 mg/ml, and 1.5 mg/ml in medium) for up to 24 hours. We determined ATP content using a luciferin-luciferase bioluminescent assay. No significant difference was found between the JP-8 jet fuel doses or time points, when compared to controls. Results were correlated with structural alterations following aerosol inhalation of JP-8. As a general observation, ultrastructural evaluation of alveolar type cells revealed an apparent increase in the number and size of surfactant secreting lamellar bodies that was JP-8 jet fuel-dose dependent. These results are similar to those observed following aerosol inhalation exposure. Thus, the lung tissue slice model appears to mimic in vivo effects of JP-8 and therefore is a useful model system for studying the mechanisms of lunginjury following JP-8 exposure.

In vitro neutralization of HCV by goat antibodies against peptides encompassing regions downstream of HVR-1 of E2 glycoprotein.

PubMed

Tabll, Ashraf A; Atef, Khaled; Bader El Din, Noha G; El Abd, Yasmine S; Salem, Ahmed; Sayed, Ahmed A; Dawood, Reham M; Omran, Moataza H; El-Awady, Mostafa K

2014-01-01

This article aims at testing several in vitro systems with various viral sources and cell lines for propagation of HCV to evaluate goat antibodies raised against three E2 epitopes in viral neutralization experiments. Four human cell lines (Huh-7, Huh-7.5, HepG2, and CaCo2) were tested using two different HCV viral sources; Genotype 4 infected sera and J6/JFH HCV cc particles. Neutralization capacity of goat Abs against conserved E2 epitopes; p412 (a.a 412-419), p517 (a.a 517-531), and p430 (a.a 430-447) were examined in the above mentioned in vitro systems. Although infection with patients' sera seems to mimic the in vitro situation, it has limited replication rates as compared with HCV cc particularly in Huh7.5 cells. Non-HCV adapted Huh-7 cells were also found susceptible for transfection with J6/JFH virus but at much slower kinetics. The results of the neutralization assay showed that anti p412 and anti p517 were highly neutralizing to HCVcc. Our data demonstrate that antibodies directed against the viral surface glycoprotein E2 reduced the infectivity of the J6/JFH virus and are promising agents for immunotherapy and HCV vaccine development.

Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

PubMed

Kawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, Tohru

2016-06-17

N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.

Novel methods of treating ovarian infertility in older and POF women, testicular infertility, and other human functional diseases.

PubMed

Bukovsky, Antonin

2015-02-25

In vitro maturation (IVM) and in vitro fertilization (IVF) technologies are facing with growing demands of older women to conceive. Although ovarian stem cells (OSCs) of older women are capable of producing in vitro fresh oocyte-like cells (OLCs), such cells cannot respond to IVM and IVF due to the lack of granulosa cells required for their maturation. Follicular renewal is also dependent on support of circulating blood mononuclear cells. They induce intermediary stages of meiosis (metaphase I chromosomal duplication and crossover, anaphase, telophase, and cytokinesis) in newly emerging ovarian germ cells, as for the first time demonstrated here, induce formation of granulosa cells, and stimulate follicular growth and development. A pretreatment of OSC culture with mononuclear cells collected from blood of a young healthy fertile woman may cause differentiation of bipotential OSCs into both developing germ and granulosa cells. A small blood volume replacement may enable treatment of ovarian infertility in vivo. The transferred mononuclear cells may temporarily rejuvenate virtually all tissues, including improvement of the function of endocrine tissues. Formation of new follicles and their development may be sufficient for IVM and IVF. The novel proposed in vitro approaches may be used as a second possibility. Infertility of human males affects almost a half of the infertility cases worldwide. Small blood volume replacement from young healthy fertile men may also be easy approach for the improvement of sperm quality in older or other affected men. In addition, body rejuvenation by small blood volume replacement from young healthy individuals of the same sex could represent a decline of in vitro methodology in favor of in vivo treatment for human functional diseases. Here we propose for the first time that blood mononuclear cells are essential for rejuvenation of those tissues, where immune system components participate in an appropriate division and differentiation of tissue stem cells. If needed, small blood volume replacement from distinct young healthy individuals could be utilized in six month intervals for repair of young altered or aged reproductive and other tissue functions. Systemic and local use of honey bee propolis tincture is an alternative option for functional rejuvenation of some tissues.

[Possibilities and limitations of fibroblast cultures in the study of animal aging].

PubMed

Van Gansen, P; Van Lerberghe, N

1987-01-01

INTRODUCTION. Aging--the effect of time--occurs in every living organism. Senescence is the last period of the lifespan, leading to death. It happens in all animals, with the exception of a few didermic species (Hydras) having a stock of embryonic cells and being immortal. The causes of animal senescence are badly known. They depend both on genetic characters (maximal lifespan of a species) and on medium factors (mean expectation of life of the animals of a species). Animal senescence could depend on cell aging: 1) by senescence and death of the differentiated cells, 2) by modified proliferation and differentiation of the stem cells of differentiated tissues, 3) by alterations in the extracellular matrices, 4) by interactions between factors 1) 2) and 3) in each tissue, 5) by interactions between the several tissues of an organism. This complexity badly impedes the experimental study of animal senescence. Normal mammal cells are aging when they are cultivated (in vitro ageing): their phenotype varies and depends on the cell generation (in vitro differentiation); the last cell-generation doesn't divide anymore and declines until death of the culture (in vitro senescence). Analysis of these artificial but well controlled systems allows an experimental approach of the proliferation, differentiation, senescence and death of the cells and of the extracellular matrix functions. Present literature upon in vitro aging of cultivated human cells is essentially made of papers where proliferation and differentiation characteristics are compared between early ("young") and late ("old") cell-generations of the cultures. FIBROBLASTIC CELLS OF THE MOUSE SKIN. This cell type has been studied in our laboratory, using different systems: 1) Primary cultures isolated from peeled skins of 19 day old mouse embryos, 2) Mouse dermis analyzed in the animals, 3) Cultivated explants of skins, 4) Serial sub-cultures of fibroblasts isolated from these explants, 5) Cells cultivated comparably on plane substrates (glass, plastic, collagen films) and on tridimensional matrices (collagen fibres). Systems 2), 3), 4) and 5) have been obtained either from 19 day old embryos or from 6 groups of animals of different ages (from 1/2 till 25 month). In primary cultures (system 1) all the cell generations have been analyzed, including the last one until death of the culture. We have shown that many characters are varying with cell-generation: cell form and cell mass, rate of DNA replication and cell division, rate of RNA transcription, nature of the accumulated and of the synthetized proteins, organization of the cytoskeletal elements, organization of the extracellular matrix, type of cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of human embryonic and induced pluripotent stem cells into urothelium.

PubMed

Osborn, Stephanie L; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H; Nolta, Jan; Kurzrock, Eric A

2014-05-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

PubMed Central

Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

2014-01-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

In vitro cell system for studying molecular mechanisms of action associated with low intensity focused ultrasound

NASA Astrophysics Data System (ADS)

Babakhanian, Meghedi; Fan, Richard E.; Mulgaonkar, Amit P.; Singh, Rahul; Culjat, Martin O.; Danesh, Shahab M.; Toro, Ligia; Grundfest, Warren; Melega, William P.

2012-03-01

Low intensity focused ultrasound (LIFU) is now being considered as a noninvasive brain therapy for clinical applications. We maintain that LIFU can efficiently deliver energy from outside the skull to target specific brain regions, effecting localized neuromodulation. However, the underlying molecular mechanisms that drive this LIFU-induced neuromodulation are not well-defined due, in part, to our lack of understanding of how particular sets of LIFU delivery parameters affect the outcome. To efficiently conduct multiple sweeps of different parameters and determine their effects, we have developed an in-vitro system to study the effects of LIFU on different types of cells grown in culture. Presently, we are evaluating how LIFU affects the ionic flux that may underlie neuronal excitation and inhibition observed in-vivo. The results of our in-vitro studies will provide a rationale for selection of optimal LIFU parameter to be used in subsequent in-vivo applications. Thus, a prototype ultrasound cell assay system has been developed to conduct these studies, and is described in this work.

Synthetic Biology for Cell-Free Biosynthesis: Fundamentals of Designing Novel In Vitro Multi-Enzyme Reaction Networks.

PubMed

Morgado, Gaspar; Gerngross, Daniel; Roberts, Tania M; Panke, Sven

Cell-free biosynthesis in the form of in vitro multi-enzyme reaction networks or enzyme cascade reactions emerges as a promising tool to carry out complex catalysis in one-step, one-vessel settings. It combines the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. Such cascades have been successfully applied to the synthesis of fine and bulk chemicals, monomers and complex polymers of chemical importance, and energy molecules from renewable resources as well as electricity. The scale of these initial attempts remains small, suggesting that more robust control of such systems and more efficient optimization are currently major bottlenecks. To this end, the very nature of enzyme cascade reactions as multi-membered systems requires novel approaches for implementation and optimization, some of which can be obtained from in vivo disciplines (such as pathway refactoring and DNA assembly), and some of which can be built on the unique, cell-free properties of cascade reactions (such as easy analytical access to all system intermediates to facilitate modeling).

The in vitro mitogenic response to intact bacteria by murine B cells does not predict in vivo susceptibility to Salmonella typhimurium.

PubMed

Elkins, K; Metcalf, E S

1986-05-01

We are interested in developing in vitro culture systems that will permit immune responses to intact Salmonella typhimurium, since these systems would have certain advantages over in vivo infection models for the characterization of the host's responding cell types. In this report, the in vitro proliferative response of nonimmune murine spleen cells to four different killed preparations of S. typhimurium, strain TML (TML), are examined. These studies show that UV-killed TML, heat-killed TML, glutaraldehyde-killed TML, and acetone-killed and dried TML, all elicit a nonspecific mitogenic spleen cell response in vitro, as does a live, avirulent, temperature-sensitive mutant of TML, TS27. This response reaches a maximum on day 2 after initiation of culture, which is similar to the time course of a conventional lipopolysaccharide (LPS) response. Unlike the LPS response, little 3H-thymidine incorporation is observed in low-density cultures (2 X 10(5) cells/well), which suggests a critical role for accessory cells. The responding cell types include, but are not necessarily limited to, the B-cell population. The response cannot be readily inhibited by polymyxin B, which binds specifically to the lipid A portion of LPS. Thus, the bacterial components required for mitogenicity are not yet definitively identified. A survey of the mitogenic responses of lymphocytes from various inbred mouse strains, including the C3H/HeJ LPS hyporesponsive strain, indicates that all B cells tested are capable of proliferating vigorously in response to intact TML, regardless of the in vivo susceptibility to virulent infection. These results also emphasize the importance of assessing the nonspecific components of the immune response when studying the specific immune response to intact S. typhimurium.

Invasion of intestinal epithelia in vitro by the parasitic nematode Trichinella spiralis.

PubMed Central

ManWarren, T; Gagliardo, L; Geyer, J; McVay, C; Pearce-Kelling, S; Appleton, J

1997-01-01

Studies of nematode establishment in intestinal niches has been hindered by the lack of a readily manipulated in vitro assay. In this report, experiments are described wherein the larval stage of the parasitic nematode Trichinella spiralis was shown to invade epithelial cell monolayers in vitro. Larvae penetrated cells and migrated through them, leaving trails of dead cells in their wake. Cells derived from five different species were susceptible to invasion, reflecting the broad host range of T. spiralis in vivo. Epithelial cells derived from large and small intestines and kidneys were susceptible. Fibroblast and muscle cells were resistant. Larvae deposited glycoprotein antigens in the cells they invaded. Although the function of these antigens is unknown, they are targeted by rat antibodies that cause T. spiralis to be expelled from the intestine. The model system described provides the means to further investigate this process as well as the mechanisms by which this parasitic nematode establishes its intestinal niche. PMID:9353069

Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

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

Li Aihua; Cheng Guangli; Zhu Genghui

Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increasedmore » in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling.« less

Modification of lymphocyte responsiveness in vitro by lambda carrageenan compared with colloidal silica and depletion of surface adherent cells.

PubMed Central

Pawelec, G; Brons, G

1978-01-01

The effects of the immunosuppressive sulphated polygalactan lambda carrageenan on in vitro models of allograft immunity were compared with the effects of removing macrophages (surface adherent and/or phagocytic cells) by established methods. Carrageenan depressed the primary mixed lymphocyte reactions, but not to the same extent as the removal of macrophages. 2-Mercaptoethanol restored the response. Secondary mixed lymphocyte reactions and responses to phytohaemaglutinin were depressed by carrageenan but not by the removal of macrophages, and in these systems 2-mercaptoethanol failed to restore the responses of carrageenan-treated cultures. In contrast, cell-mediated cytolysis by presensitized lymphocytes was not affected by carrageenan or by colloidal silica. Carrageenan depressed cell-mediated cytolysis only if it was present during the sensitization of the effector cells. We conclude that carrageenan can have two dose-related effects in vitro: one on the macrophage and one on the responding lymphocyte. PMID:207475

Cytotoxicity assessment of antibiofouling compounds and by-products in marine bivalve cell cultures.

PubMed

Domart-Coulon, I; Auzoux-Bordenave, S; Doumenc, D; Khalanski, M

2000-06-01

Short-term primary cell cultures were derived from adult marine bivalve tissues: the heart of oyster Crassostrea gigas and the gill of clam Ruditapes decussatus. These cultures were used as experimental in vitro models to assess the acute cytotoxicity of an organic molluscicide, Mexel-432, used in antibiofouling treatments in industrial cooling water systems. A microplate cell viability assay, based on the enzymatic reduction of tetrazolium dye (MTT) in living bivalve cells, was adapted to test the cytotoxicity of this compound: in both in vitro models, toxicity thresholds of Mexel-432 were compared to those determined in vivo with classic acute toxicity tests. The clam gill cell model was also used to assess the cytotoxicity of by-products of chlorination, a major strategy of biofouling control in the marine environment. The applications and limits of these new in vitro models for monitoring aquatic pollutants were discussed, in reference with the standardized Microtox test.

Quinazoline clubbed 1,3,5-triazine derivatives as VEGFR2 kinase inhibitors: design, synthesis, docking, in vitro cytotoxicity and in ovo antiangiogenic activity.

PubMed

Pathak, Prateek; Shukla, Parjanya Kumar; Kumar, Vikas; Kumar, Ankit; Verma, Amita

2018-04-16

A series of quinazoline clubbed 1,3,5-triazine derivatives (QCT) were synthesized and evaluated for their in vitro anticancer activity against HeLa (human cervical cancer), MCF-7 (human breast cancer cell), HL-60 (human promyelocytic leukemia cell), HepG2 (human Hepatocellular carcinoma cell), and one normal cell line HFF (human foreskin fibroblasts). In vitro assay result encouraged to further move towards in ovo anticancer evaluation using chick embryo. The series of QCT derivatives showed higher anticancer and antiangiogenic activity against HeLa and MCF-7 cell lines. In the series, synthetic molecule 8d, 8l, and 8m displayed significant activity. Further, these results substantiated by docking study on VGFR2. SAR study concluded that the potency of drugs depends on the nature of aliphatic substitution and the heterocyclic ring system.

A Novel Model System to Examine Agents Used in Breast Cancer Therapy.

DTIC Science & Technology

1996-07-01

DNA replication (DNA synthesome) isolated from MDA MB 468 human breast cancer cells, human breast tumor tissue and human breast tumor cell xenografts In the presence of the viral large T-antigen and simian virus 40 (SV40) origin sequences, the DNA synthesome executes all of the steps required for the in vitro replication of the SV40 genome. Furthermore, the DNA synthesome isolated from human breast cancer cells possesses a lower fidelity for DNA synthesis in vitro than the synthesome purified from a non-malignant breast cell line. Our studies indicate that the following

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

PubMed

Mayrhofer, Corina; Krieger, Sigurd; Allmaier, Günter; Kerjaschki, Dontscho

2006-01-01

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

The fertilization ability and developmental competence of bovine oocytes grown in vitro

PubMed Central

MAKITA, Miho; UEDA, Mayuko; MIYANO, Takashi

2016-01-01

In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4−0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts. PMID:27151093

The fertilization ability and developmental competence of bovine oocytes grown in vitro.

PubMed

Makita, Miho; Ueda, Mayuko; Miyano, Takashi

2016-08-25

In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.

Multifunctional cell-culture platform for aligned cell sheet monitoring, transfer printing, and therapy.

PubMed

Kim, Seok Joo; Cho, Hye Rim; Cho, Kyoung Won; Qiao, Shutao; Rhim, Jung Soo; Soh, Min; Kim, Taeho; Choi, Moon Kee; Choi, Changsoon; Park, Inhyuk; Hwang, Nathaniel S; Hyeon, Taeghwan; Choi, Seung Hong; Lu, Nanshu; Kim, Dae-Hyeong

2015-03-24

While several functional platforms for cell culturing have been proposed for cell sheet engineering, a soft integrated system enabling in vitro physiological monitoring of aligned cells prior to their in vivo applications in tissue regeneration has not been reported. Here, we present a multifunctional, soft cell-culture platform equipped with ultrathin stretchable nanomembrane sensors and graphene-nanoribbon cell aligners, whose system modulus is matched with target tissues. This multifunctional platform is capable of aligning plated cells and in situ monitoring of cellular physiological characteristics during proliferation and differentiation. In addition, it is successfully applied as an in vitro muscle-on-a-chip testing platform. Finally, a simple but high-yield transfer printing mechanism is proposed to deliver cell sheets for scaffold-free, localized cell therapy in vivo. The muscle-mimicking stiffness of the platform allows the high-yield transfer printing of multiple cell sheets and results in successful therapies in diseased animal models. Expansion of current results to stem cells will provide unique opportunities for emerging classes of tissue engineering and cell therapy technologies.

Establishment of an in vitro cell line experimental system for the study of inhalational anesthetic mechanisms.

PubMed

Nagamoto, Seiji; Iijima, Norio; Ishii, Hirotaka; Takumi, Ken; Higo, Shimpei; Aikawa, Satoko; Anzai, Megumi; Matsuo, Izumi; Nakagawa, Shinji; Takashima, Naoyuki; Shigeyoshi, Yasufumi; Sakamoto, Atsuhiro; Ozawa, Hitoshi

2016-05-04

General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia. Here, we established an in vitro experimental system using a cell line to investigate the mechanisms underlying anesthetic action. Development of the system comprised two steps: first, we developed a system for application of inhalational anesthetics and incubation; next, we established cultures of anesthetic-responsive cells expressing mPer2 promoter-dLuc. GT1-7 cells, derived from the mouse hypothalamus, responded to sevoflurane by reversibly decreasing mPer2-promoter-driven bioluminescence. Interestingly, the suppression of bioluminescence was found only in the serum-starved GT1-7 cells, which showed neuron-like morphology, but not in growing cells, suggesting that neuron-like characteristics are required for anesthetic effects in GT1-7 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4.

PubMed Central

Wu, S Y; Kershnar, E; Chiang, C M

1998-01-01

TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs. PMID:9687514

In vitro effects of Epidiferphane™ on adult human neural progenitor cells

USDA-ARS?s Scientific Manuscript database

Neural stem cells have the capacity to respond to their environment, migrate to the injury site and generate functional cell types, and thus they hold great promise for cell therapies. In addition to representing a source for central nervous system (CNS) repair, neural stem and progenitor cells als...

Comparison of Chemical Binding to Recombinant Fathead minnow and Human Estrogen Receptor alpha (ERα) in Whole Cell and Cell-Free Assay Systems.

EPA Science Inventory

Our objectives were to assess whether binding of chemicals differs significantly between recombinant estrogen receptors from fathead minnow (fhERα) and human (hERα) and to evaluate the performance of these receptors using two different in vitro assay systems: a COS whole cell bin...

In vitro techniques for the assessment of neurotoxicity.

PubMed Central

Harry, G J; Billingsley, M; Bruinink, A; Campbell, I L; Classen, W; Dorman, D C; Galli, C; Ray, D; Smith, R A; Tilson, H A

1998-01-01

Risk assessment is a process often divided into the following steps: a) hazard identification, b) dose-response assessment, c) exposure assessment, and d) risk characterization. Regulatory toxicity studies usually are aimed at providing data for the first two steps. Human case reports, environmental research, and in vitro studies may also be used to identify or to further characterize a toxic hazard. In this report the strengths and limitations of in vitro techniques are discussed in light of their usefulness to identify neurotoxic hazards, as well as for the subsequent dose-response assessment. Because of the complexity of the nervous system, multiple functions of individual cells, and our limited knowledge of biochemical processes involved in neurotoxicity, it is not known how well any in vitro system would recapitulate the in vivo system. Thus, it would be difficult to design an in vitro test battery to replace in vivo test systems. In vitro systems are well suited to the study of biological processes in a more isolated context and have been most successfully used to elucidate mechanisms of toxicity, identify target cells of neurotoxicity, and delineate the development and intricate cellular changes induced by neurotoxicants. Both biochemical and morphological end points can be used, but many of the end points used can be altered by pharmacological actions as well as toxicity. Therefore, for many of these end points it is difficult or impossible to set a criterion that allows one to differentiate between a pharmacological and a neurotoxic effect. For the process of risk assessment such a discrimination is central. Therefore, end points used to determine potential neurotoxicity of a compound have to be carefully selected and evaluated with respect to their potential to discriminate between an adverse neurotoxic effect and a pharmacologic effect. It is obvious that for in vitro neurotoxicity studies the primary end points that can be used are those affected through specific mechanisms of neurotoxicity. For example, in vitro systems may be useful for certain structurally defined compounds and mechanisms of toxicity, such as organophosphorus compounds and delayed neuropathy, for which target cells and the biochemical processes involved in the neurotoxicity are well known. For other compounds and the different types of neurotoxicity, a mechanism of toxicity needs to be identified first. Once identified, by either in vivo or in vitro methods, a system can be developed to detect and to evaluate predictive ability for the type of in vivo neurotoxicity produced. Therefore, in vitro tests have their greatest potential in providing information on basic mechanistic processes in order to refine specific experimental questions to be addressed in the whole animal. Images Figure 1 PMID:9539010

DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing.

PubMed

Simara, Pavel; Tesarova, Lenka; Rehakova, Daniela; Matula, Pavel; Stejskal, Stanislav; Hampl, Ales; Koutna, Irena

2017-03-21

Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.

Integrin receptor involvement in actin cable formation in an in vitro model of events associated with wound contraction.

PubMed

Stephens, P; Genever, P G; Wood, E J; Raxworthy, M J

1997-01-01

Actin cables have been reported to act in vivo as contractile 'purse strings' capable of closing embryonic wounds through generation of circumferential tension. Furthermore, their involvement in wounds within in vitro model systems suggests that actin cable contraction may be an important mechanism involved in the process of wound closure. The aim of this study therefore, was to investigate the appearance of actin cables in a contracting fibroblast populated collagen lattice, an in vitro model of events associated with wound contraction. Utilising this in vitro model, the time-course of actin cable production was investigated and the involvement of integrin receptors analysed using immunofluorescent labelling techniques. Over a period of hours distinct cellular cable-like structures developed at the edges of collagen lattices coinciding with the onset of contraction. Cellular organisation within the cable was evident as was polymerisation of actin microfilaments into elongated stress fibres forming a continuous cell-cell 'actin cable' around the circumference of the lattice. Immunolocalisation demonstrated that integrin receptor subunits beta 1 and alpha 2 but not alpha 5 were involved in apparent intimate cell-cell contact between juxtaposed fibroblasts within this actin cable. This study demonstrates the involvement of integrin receptors in actin cable formation within collagen lattice systems undergoing reorganisation. Such integrin involvement may enable participating cells to respond to the tensional status of their surrounding environment and via cell-cell communication, to permit a co-ordinated contraction of the cable. It is concluded that integrin receptor involvement in active actin cable contraction may be involved in the process of wound contraction.

Aptamer redesigned tRNA is nonfunctional and degraded in cells

PubMed Central

LEE, DENNIS; MCCLAIN, WILLIAM H.

2004-01-01

An RNA aptamer derived from tRNAGln isolated in vitro and a rationally redesigned tRNAGln were used to address the relationship between structure and function of tRNAGln aminoacylation in Escherichia coli. Two mutant tRNAGln sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNAGln in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm. Both mutants were tested in a tRNAGln knockout strain of E. coli, but neither supported knockout cell growth. It was later found that both mutant tRNAs were present in very low amounts in the cell. These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro. PMID:14681579

In vitro vascularization of a combined system based on a 3D printing technique.

PubMed

Zhao, Xinru; Liu, Libiao; Wang, Jiayin; Xu, Yufan; Zhang, Weiming; Khang, Gilson; Wang, Xiaohong

2016-10-01

A vital challenge in complex organ manufacturing is to vascularize large combined tissues. The aim of this study is to vascularize in vitro an adipose-derived stem cell (ADSC)/fibrin/collagen incorporated three-dimensional (3D) poly(d,l-lactic-co-glycolic acid) (PLGA) scaffold (10 × 10 × 10 mm 3 ) with interconnected channels. A low-temperature 3D printing technique was employed to build the PLGA scaffold. A step-by-step cocktail procedure was designed to engage or steer the ADSCs in the PLGA channels towards both endothelial and smooth muscle cell lineages. The combined system had sufficient mechanical properties to support the cell/fibrin/collagen hydrogel inside the predefined PLGA channels. The ADSCs encapsulated in the fibrin/collagen hydrogel differentiated to endothelial and smooth muscle cell lineage, respectively, corresponding to their respective locations in the construct and formed vascular-like structures. This technique allows in vitro vascularization of the predefined PLGA channels and provides a choice for complex organ manufacture. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

In vivo osteogenic differentiation of stem cells inside compartmentalized capsules loaded with co-cultured endothelial cells.

PubMed

Correia, Clara R; Santos, Tírcia C; Pirraco, Rogério P; Cerqueira, Mariana T; Marques, Alexandra P; Reis, Rui L; Mano, João F

2017-04-15

Capsules coated with polyelectrolytes and co-encapsulating adipose stem (ASCs) and endothelial (ECs) cells with surface modified microparticles are developed. Microparticles and cells are freely dispersed in a liquified core, responsible to maximize the diffusion of essential molecules and allowing the geometrical freedom for the autonomous three-dimensional (3D) organization of cells. While the membrane wraps all the instructive cargo elements within a single structure, the microparticles provide a solid 3D substrate for the encapsulated cells. Our hypothesis is that inside this isolated biomimetic 3D environment, ECs would lead ASCs to differentiate into the osteogenic lineage to ultimately generate a mineralized tissue in vivo. For that, capsules encapsulating only ASCs (MONO capsules) or co-cultured with ECs (CO capsules) are subcutaneously implanted in nude mice up to 6weeks. Capsules implanted immediately after production or after 21days of in vitro osteogenic stimulation are tested. The most valuable outcome of the present study is the mineralized tissue in CO capsules without in vitro pre-differentiation, with similar levels compared to the pre-stimulated capsules in vitro. We believe that the proposed bioencapsulation strategy is a potent self-regulated system, which might find great applicability in bone tissue engineering. The diffusion efficiency of essential molecules for cell survival is a main issue in cell encapsulation. Former studies reported the superior biological outcome of encapsulated cells within liquified systems. However, most cells used in TE are anchorage-dependent, requiring a solid substrate to perform main cellular processes. We hypothesized that liquified capsules encapsulating microparticles are a promising attempt. Inspired by the multiphenotypic cellular environment of bone, we combine the concept of liquified capsules with co-cultures of stem and endothelial cells. After implantation, results show that co-cultured capsules without in vitro stimulation were able to form a mineralized tissue in vivo. We believe that the present ready-to-use TE strategy requiring minimum in vitro manipulation will find great applicability in bone tissue engineering. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Microfluidic co-culture devices to assess penetration of nanoparticles into cancer cell mass.

PubMed

Jarvis, Maria; Arnold, Michael; Ott, Jenna; Pant, Kapil; Prabhakarpandian, Balabhaskar; Mitragotri, Samir

2017-09-01

In vitro and in vivo assessment of safety and efficacy are the essential first steps in developing nanoparticle-based therapeutic systems. However, it is often challenging to use the knowledge gained from in vitro studies to predict the outcome of in vivo studies since the complexity of the in vivo environment, including the existence of flow and a multicellular environment, is often lacking in traditional in vitro models. Here, we describe a microfluidic co-culture model comprising 4T1 breast cancer cells and EA.hy926 endothelial cells under physiological flow conditions and its utilization to assess the penetration of therapeutic nanoparticles from the vascular compartment into a cancerous cell mass. Camptothecin nanocrystals (∼310 nm in length), surface-functionalized with PEG or folic acid, were used as a test nanocarrier. Camptothecin nanocrystals exhibited only superficial penetration into the cancerous cell mass under fluidic conditions, but exhibited cytotoxicity throughout the cancerous cell mass. This likely suggests that superficially penetrated nanocrystals dissolve at the periphery and lead to diffusion of molecular camptothecin deep into the cancerous cell mass. The results indicate the potential of microfluidic co-culture devices to assess nanoparticle-cancerous cell interactions, which are otherwise difficult to study using standard in vitro cultures.

Structure-Activity–Dependent Regulation of Cell Communication by Perfluorinated Fatty Acids using in Vivo and in Vitro Model Systems

PubMed Central

Upham, Brad L.; Park, Joon-Suk; Babica, Pavel; Sovadinova, Iva; Rummel, Alisa M.; Trosko, James E.; Hirose, Akihiko; Hasegawa, Ryuichi; Kanno, Jun; Sai, Kimie

2009-01-01

Background Perfluoroalkanoates, [e.g., perfluorooctanoate (PFOA)], are known peroxisome proliferators that induce hepatomegaly and hepatocarcinogenesis in rodents, and are classic non-genotoxic carcinogens that inhibit in vitro gap-junctional intercellular communication (GJIC). This inhibition of GJIC is known to be a function of perfluorinated carbon lengths ranging from 7 to 10. Objectives The aim of this study was to determine if the inhibition of GJIC by PFOA but not perfluoropentanoate (PFPeA) observed in F344 rat liver cells in vitro also occurs in F344 rats in vivo and to determine mechanisms of PFOA dysregulation of GJIC using in vitro assay systems. Methods We used an incision load/dye transfer technique to assess GJIC in livers of rats exposed to PFOA and PFPeA. We used in vitro assays with inhibitors of cell signaling enzymes and antioxidants known to regulate GJIC to identify which enzymes regulated PFOA-induced inhibition of GJIC. Results PFOA inhibited GJIC and induced hepatomegaly in rat livers, whereas PFPeA had no effect on either end point. Serum biochemistry of liver enzymes indicated no cytotoxic response to these compounds. In vitro analysis of mitogen-activated protein kinase (MAPK) indicated that PFOA, but not PFPeA, can activate the extracellular receptor kinase (ERK). Inhibition of GJIC, in vitro, by PFOA depended on the activation of both ERK and phosphatidylcholine-specific phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism. Conclusions The in vitro analysis of GJIC, an epigenetic marker of tumor promoters, can also predict the in vivo activity of PFOA, which dysregulated GJIC via ERK and PC-PLC. PMID:19440492

3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

PubMed

Vidal, Sarah E Lightfoot; Tamamoto, Kasey A; Nguyen, Hanh; Abbott, Rosalyn D; Cairns, Dana M; Kaplan, David L

2018-04-24

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

Comparison of human chordoma cell-kill for 290 MeV/n carbon ions versus 70 MeV protons in vitro

PubMed Central

2013-01-01

Background While the pace of commissioning of new charged particle radiation therapy facilities is accelerating worldwide, biological data pertaining to chordomas, theoretically and clinically optimally suited targets for particle radiotherapy, are still lacking. In spite of the numerous clinical reports of successful treatment of these malignancies with this modality, the characterization of this malignancy remains hampered by its characteristic slow cell growth, particularly in vitro. Methods Cellular lethality of U-CH1-N cells in response to different qualities of radiation was compared with immediate plating after radiation or as previously reported using the multilayered OptiCell™ system. The OptiCell™ system was used to evaluate cellular lethality over a broad dose-depth deposition range of particle radiation to anatomically mimic the clinical setting. Cells were irradiated with either 290 MeV/n accelerated carbon ions or 70 MeV accelerated protons and photons and evaluated through colony formation assays at a single position or at each depth, depending on the system. Results There was a cell killing of approximately 20–40% for all radiation qualities in the OptiCell™ system in which chordoma cells are herein described as more radiation sensitive than regular colony formation assay. The relative biological effectiveness values were, however, similar in both in vitro systems for any given radiation quality. Relative biological effectiveness values of proton was 0.89, of 13–20 keV/μm carbon ions was 0.85, of 20–30 keV/μm carbon ions was 1.27, and >30 keV/μm carbon ions was 1.69. Carbon-ions killed cells depending on both the dose and the LET, while protons depended on the dose alone in the condition of our study. This is the first report and characterization of a direct comparison between the effects of charged particle carbon ions versus protons for a chordoma cell line in vitro. Our results support a potentially superior therapeutic value of carbon particle irradiation in chordoma patients. Conclusion Carbon ion therapy may have an advantage for chordoma radiotherapy because of higher cell-killing effect with high LET doses from biological observation in this study. PMID:23587329

Methodological uncertainty in quantitative prediction of human hepatic clearance from in vitro experimental systems.

PubMed

Hallifax, D; Houston, J B

2009-03-01

Mechanistic prediction of unbound drug clearance from human hepatic microsomes and hepatocytes correlates with in vivo clearance but is both systematically low (10 - 20 % of in vivo clearance) and highly variable, based on detailed assessments of published studies. Metabolic capacity (Vmax) of commercially available human hepatic microsomes and cryopreserved hepatocytes is log-normally distributed within wide (30 - 150-fold) ranges; Km is also log-normally distributed and effectively independent of Vmax, implying considerable variability in intrinsic clearance. Despite wide overlap, average capacity is 2 - 20-fold (dependent on P450 enzyme) greater in microsomes than hepatocytes, when both are normalised (scaled to whole liver). The in vitro ranges contrast with relatively narrow ranges of clearance among clinical studies. The high in vitro variation probably reflects unresolved phenotypical variability among liver donors and practicalities in processing of human liver into in vitro systems. A significant contribution from the latter is supported by evidence of low reproducibility (several fold) of activity in cryopreserved hepatocytes and microsomes prepared from the same cells, between separate occasions of thawing of cells from the same liver. The large uncertainty which exists in human hepatic in vitro systems appears to dominate the overall uncertainty of in vitro-in vivo extrapolation, including uncertainties within scaling, modelling and drug dependent effects. As such, any notion of quantitative prediction of clearance appears severely challenged.

When nano meets stem: the impact of nanotechnology in stem cell biology.

PubMed

Kaur, Savneet; Singhal, Barkha

2012-01-01

Nanotechnology and biomedical treatments using stem cells are among the latest conduits of biotechnological research. Even more recently, scientists have begun finding ways to mate these two specialties of science. The advent of nanotechnology has paved the way for an explicit understanding of stem cell therapy in vivo and by recapitulation of such in vivo environments in the culture, this technology seems to accommodate a great potential in providing new vistas to stem cell research. Nanotechnology carries in its wake, the development of highly stable, efficient and specific gene delivery systems for both in vitro and in vivo genetic engineering of stem cells, use of nanoscale systems (such as microarrays) for investigation of gene expression in stem cells, creation of dynamic three-dimensional nano-environments for in vitro and in vivo maintenance and differentiation of stem cells and development of extremely sensitive in vivo detection systems to gain insights into the mechanisms of stem cell differentiation and apoptosis in different disease models. The present review presents an overview of the current applications and future prospects for the use of nanotechnology in stem cell biology. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Evaluation of air-liquid interface exposure systems for in vitro assessment of airborne pollutants

EPA Science Inventory

Exposure of cells to airborne pollutants at the air-liquid interface (ALI) is a more realistic approach than exposures of submerged cells. The published literature, however, describes irreproducible and/or unrealistic experimental conditions using ALI systems. We have compared fi...

Applications of human umbilical cord blood cells in central nervous system regeneration.

PubMed

Herranz, Antonio S; Gonzalo-Gobernado, Rafael; Reimers, Diana; Asensio, Maria J; Rodríguez-Serrano, Macarena; Bazán, Eulalia

2010-03-01

In recent decades, there has been considerable amount of information about embryonic stem cells (ES). The dilemma facing scientists interested in the development and use of human stem cells in replacement therapies is the source of these cells, i.e. the human embryo. There are many ethical and moral problems related to the use of these cells. Hematopoietic stem cells from umbilical cord blood have been proposed as an alternative source of embryonic stem cells. After exposure to different agents, these cells are able to express antigens of diverse cellular lineages, including the neural type. The In vitro manipulation of human umbilical cord blood (hUCB) cells has shown their stem capacity and plasticity. These cells are easily accessible, In vitro amplifiable, well tolerated by the host, and with more primitive molecular characteristics that give them great flexibility. Overall, these properties open a promising future for the use of hUCB in regenerative therapies for the Central Nervous System (CNS). This review will focus on the available literature concerning umbilical cord blood cells as a therapeutic tool for the treatment of neurodegenerative diseases.

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.

A Novel Source of Cultured Podocytes

PubMed Central

Da Sacco, Stefano; Lemley, Kevin V.; Sedrakyan, Sargis; Zanusso, Ilenia; Petrosyan, Astgik; Peti-Peterdi, Janos; Burford, James; De Filippo, Roger E.; Perin, Laura

2013-01-01

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies. PMID:24349133

A role for Lin28 in primordial germ cell development and germ cell malignancy

PubMed Central

West, Jason A.; Viswanathan, Srinivas R.; Yabuuchi, Akiko; Cunniff, Kerianne; Takeuchi, Ayumu; Park, In-Hyun; Sero, Julia E.; Zhu, Hao; Perez-Atayde, Antonio; Frazier, A. Lindsay; Surani, M. Azim; Daley, George Q.

2009-01-01

The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwarts efforts to investigate molecular mechanisms of germ cell specification. Stella marks the minute founder population of the germ lineage1,2. Here we differentiate mouse embryonic stem cells (ESCs) carrying a Stella transgenic reporter into putative PGCs in vitro. The Stella+ cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of Stella+ cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing3-6, is essential for proper PGC development. We further show that Blimp1, a let-7 target and a master regulator of PGC specification7-9, can rescue the effect of Lin28-deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Over-expression of Lin28 promotes formation of Stella+ cells in vitro and PGCs in chimeric embryos, and is associated with human germ cell tumours. The differentiation of putative PGCs from ESCs in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ cell development and malignancy. PMID:19578360

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.

Evaluation of in vitro spermatogenesis system effectiveness to study genes behavior: monitoring the expression of the testis specific 10 (Tsga10) gene as a model.

PubMed

Miryounesi, Mohammad; Nayernia, Karim; Mobasheri, Maryam Beigom; Dianatpour, Mahdi; Oko, Richard; Savad, Shahram; Modarressi, Mohammad Hossein

2014-10-01

In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (mESC) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. mESCs were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th, and 25th days of the culture and were subjected to expression analysis of a panel of germ cell specific genes. Expression of Tsga10 in RNA and protein levels was then analyzed. Transition from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This finding is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed forms of the related protein was similar to those in vivo as well. Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.

Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens.

PubMed

March, Sandra; Ramanan, Vyas; Trehan, Kartik; Ng, Shengyong; Galstian, Ani; Gural, Nil; Scull, Margaret A; Shlomai, Amir; Mota, Maria M; Fleming, Heather E; Khetani, Salman R; Rice, Charles M; Bhatia, Sangeeta N

2015-12-01

The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.

Evaluation of Iodine Bioavailability in Seaweed Using in Vitro Methods.

PubMed

Domínguez-González, M Raquel; Chiocchetti, Gabriela M; Herbello-Hermelo, Paloma; Vélez, Dinoraz; Devesa, Vicenta; Bermejo-Barrera, Pilar

2017-09-27

Due to the high levels of iodine present in seaweed, the ingestion of a large amount of this type of food can produce excessive intake of iodine. However, the food after ingestion undergoes different chemistry and physical processes that can modify the amount of iodine that reaches the systemic circulation (bioavailability). Studies on the bioavailability of iodine from food are scarce and indicate that the bioavailable amount is generally lower than ingested. Iodine in vitro bioavailability estimation from different commercialized seaweed has been studied using different in vitro approaches (solubility, dialyzability, and transport and uptake by intestinal cells). Results indicate that iodine is available after gastrointestinal digestion for absorption (bioaccessibility: 49-82%), kombu being the seaweed with the highest bioaccessibility. The incorporation of dialysis cell cultures to elucidate bioavailability modifies the estimation of the amount of iodine that may reach the systemic circulation (dialysis, 5-28%; cell culture, ≤3%). The paper discusses advantages and drawbacks of these methodologies for iodine bioavailability in seaweed.

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System.

PubMed

Hunsberger, Joshua G; Efthymiou, Anastasia G; Malik, Nasir; Behl, Mamta; Mead, Ivy L; Zeng, Xianmin; Simeonov, Anton; Rao, Mahendra

2015-08-15

There is great need to develop more predictive drug discovery tools to identify new therapies to treat diseases of the central nervous system (CNS). Current nonpluripotent stem cell-based models often utilize non-CNS immortalized cell lines and do not enable the development of personalized models of disease. In this review, we discuss why in vitro models are necessary for translational research and outline the unique advantages of induced pluripotent stem cell (iPSC)-based models over those of current systems. We suggest that iPSC-based models can be patient specific and isogenic lines can be differentiated into many neural cell types for detailed comparisons. iPSC-derived cells can be combined to form small organoids, or large panels of lines can be developed that enable new forms of analysis. iPSC and embryonic stem cell-derived cells can be readily engineered to develop reporters for lineage studies or mechanism of action experiments further extending the utility of iPSC-based systems. We conclude by describing novel technologies that include strategies for the development of diversity panels, novel genomic engineering tools, new three-dimensional organoid systems, and modified high-content screens that may bring toxicology into the 21st century. The strategic integration of these technologies with the advantages of iPSC-derived cell technology, we believe, will be a paradigm shift for toxicology and drug discovery efforts.

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Electrophysiological properties of neurons derived from human stem cells and iNeurons in vitro.

PubMed

Halliwell, Robert F

2017-06-01

Functional studies of neurons have traditionally used nervous system tissues from a variety of non-human vertebrate and invertebrate species, even when the focus of much of this research has been directed at understanding human brain function. Over the last decade, the identification and isolation of human stem cells from embryonic, tissue (or adult) and induced pluripotent stem cells (iPSCs) has revolutionized the availability of human neurons for experimental studies in vitro. In addition, the direct conversion of terminally differentiated fibroblasts into Induced neurons (iN) has generated great excitement because of the likely value of such human stem cell derived neurons (hSCNs) and iN cells in drug discovery, neuropharmacology, neurotoxicology and regenerative medicine. This review addresses the current state of our knowledge of functional receptors and ion channels expressed in neurons derived from human stem cells and iNeurons and identifies gaps and questions that might be investigated in future studies; it focusses almost exclusively on what is known about the electrophysiological properties of neurons derived from human stem cells and iN cells in vitro with an emphasis on voltage and ligand gated ion channels, since these mediate synaptic signalling in the nervous system and they are at the heart of neuropharmacology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail.

PubMed

Berger, Benjamin; Donzelli, Massimiliano; Maseneni, Swarna; Boess, Franziska; Roth, Adrian; Krähenbühl, Stephan; Haschke, Manuel

2016-01-01

Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suitability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least four-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20 μM rifampicin, mRNA increased 3- to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and four-fold (CYP3A4) for HepG2. In 3D-PHH at least a two-fold increase in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model. Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro . HepG2 cells are less suited to assess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro .

Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail

PubMed Central

Berger, Benjamin; Donzelli, Massimiliano; Maseneni, Swarna; Boess, Franziska; Roth, Adrian; Krähenbühl, Stephan; Haschke, Manuel

2016-01-01

Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suitability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least four-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20 μM rifampicin, mRNA increased 3- to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and four-fold (CYP3A4) for HepG2. In 3D-PHH at least a two-fold increase in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model. Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to assess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro. PMID:27917125

In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines

PubMed Central

Tomankova, Katerina; Polakova, Katerina; Pizova, Klara; Binder, Svatopluk; Havrdova, Marketa; Kolarova, Mary; Kriegova, Eva; Zapletalova, Jana; Malina, Lukas; Horakova, Jana; Malohlava, Jakub; Kolokithas-Ntoukas, Argiris; Bakandritsos, Aristides; Kolarova, Hana; Zboril, Radek

2015-01-01

One of the promising strategies for improvement of cancer treatment is based on magnetic drug delivery systems, thus avoiding side effects of standard chemotherapies. Superparamagnetic iron oxide (SPIO) nanoparticles have ideal properties to become a targeted magnetic drug delivery contrast probes, named theranostics. We worked with SPIO condensed colloidal nanocrystal clusters (MagAlg) prepared through a new soft biomineralization route in the presence of alginate as the polymeric shell and loaded with doxorubicin (DOX). The aim of this work was to study the in vitro cytotoxicity of these new MagAlg–DOX systems on mouse fibroblast and breast carcinoma cell lines. For proper analysis and understanding of cell behavior after administration of MagAlg–DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized. It was found that the cytotoxic effect of MagAlg–DOX system is delayed compared to free DOX in both cell lines. This was attributed to the different mechanism of internalization of DOX and MagAlg–DOX into the cells, together with the fact that the drug is strongly bound on the drug nanocarriers. We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line. This is a first comprehensive study on the cytotoxic effect of DOX-loaded SPIO compared with free DOX on healthy and cancer cell lines, as well as on the induced changes in gene expression. PMID:25673990

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold.

PubMed

Bouet, G; Cruel, M; Laurent, C; Vico, L; Malaval, L; Marchat, D

2015-05-15

An engineered three dimensional (3D) in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D) models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a "proof-of-concept" for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

Implementing oxygen control in chip-based cell and tissue culture systems.

PubMed

Oomen, Pieter E; Skolimowski, Maciej D; Verpoorte, Elisabeth

2016-09-21

Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier

PubMed Central

2013-01-01

Background Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Methods Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Results Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Conclusions Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery. PMID:23773766

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

PubMed

Watson, P Marc D; Paterson, Judy C; Thom, George; Ginman, Ulrika; Lundquist, Stefan; Webster, Carl I

2013-06-18

Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Controlled release of 18-β-glycyrrhetic acid by nanodelivery systems increases cytotoxicity on oral carcinoma cell line

NASA Astrophysics Data System (ADS)

Cacciotti, Ilaria; Chronopoulou, Laura; Palocci, Cleofe; Amalfitano, Adriana; Cantiani, Monica; Cordaro, Massimo; Lajolo, Carlo; Callà, Cinzia; Boninsegna, Alma; Lucchetti, Donatella; Gallenzi, Patrizia; Sgambato, Alessandro; Nocca, Giuseppina; Arcovito, Alessandro

2018-07-01

The topical treatment for oral mucosal diseases is often based on products optimized for dermatologic applications; consequently, a lower therapeutic effect may be present. 18-β-glycyrrhetic acid (GA) is extracted from Glycirrhiza glabra. The first aim of this study was to test the cytotoxicity of GA on PE/CA-PJ15 cells. The second aim was to propose and test two different delivery systems, i.e. nanoparticles and fibers, to guarantee a controlled release of GA in vitro. We used chitosan and poly(lactic-co-glycolic) acid based nanoparticles and polylactic acid fibers. We tested both delivery systems in vitro on PE/CA-PJ15 cells and on normal human gingival fibroblasts (HGFs). The morphology of GA-loaded nanoparticles (GA-NPs) and fibers (GA-FBs) was investigated by electron microscopy and dynamic light scattering; GA release kinetics was studied spectrophotometrically. MTT test was used to assess GA cytotoxicity on both cancer and normal cells. Cells were exposed to different concentrations of GA (20–500 μmol l‑1) administered as free GA (GA-f), and to GA-NPs or GA-FBs. ROS production was evaluated using dichlorodihydrofluorescein as a fluorescent probe. Regarding the cytotoxic effect of GA on PE/CA-PJ15 cells, the lowest TC50 value was 200 μmol l‑1 when GA was added as GA-NPs. No cytotoxic effects were observed when GA was administered to HGFs. N-acetyl Cysteine reduced mortality induced by GA-f in PE/CA-PJ15 cells. The specific effect of GA on PE/CA-PJ15 cells is mainly due to the different sensitivity of cancer cells to ROS over-production; GA-NPs and GA-FBs formulations increase, in vitro, this toxic effect on oral cancer cells.

Human kidney proximal tubule cells are vulnerable to the effects of Rauwolfia serpentina.

PubMed

Mossoba, Miriam E; Flynn, Thomas J; Vohra, Sanah; Wiesenfeld, Paddy L; Sprando, Robert L

2015-12-01

Rauwolfia serpentina (or Snake root plant) is a botanical dietary supplement marketed in the USA for maintaining blood pressure. Very few studies have addressed the safety of this herb, despite its wide availability to consumers. Its reported pleiotropic effects underscore the necessity for evaluating its safety. We used a human kidney cell line to investigate the possible negative effects of R. serpentina on the renal system in vitro, with a specific focus on the renal proximal tubules. We evaluated cellular and mitochondrial toxicity, along with a variety of other kidney-specific toxicology biomarkers. We found that R. serpentina was capable of producing highly detrimental effects in our in vitro renal cell system. These results suggest more studies are needed to investigate the safety of this dietary supplement in both kidney and other target organ systems.

Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells (ASC) using an in vitro three-dimensional micromass culture system

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

Xu, Yue; Balooch, Guive; Chiou, Michael

2007-07-27

Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitromore » model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.« less

Prediction of delivery of organic aerosols onto air-liquid interface cells in vitro using an electrostatic precipitator.

PubMed

Yu, Zechen; Jang, Myoseon; Sabo-Attwood, Tara; Robinson, Sarah E; Jiang, Huanhuan

2017-08-01

To better characterize biological responses to atmospheric organic aerosols, the efficient delivery of aerosol to in vitro lung cells is necessary. In this study, chamber generated secondary organic aerosol (SOA) entered the commercialized exposure chamber (CULTEX® Radial Flow System Compact) where it interfaced with an electrostatic precipitator (ESP) (CULTEX® Electrical Deposition Device) and then deposited on a particle collection plate. This plate contained human lung cells (BEAS-2B) that were cultured on a membrane insert to produce an air-liquid interface (ALI). To augment in vitro assessment using the ESP exposure device, the particle dose was predicted for various sampling parameters such as particle size, ESP deposition voltage, and sampling flowrate. The dose model was evaluated against the experimental measured mass of collected airborne particles. The high flowrate used in this study increased aerosol dose but failed to achieve cell stability. For example, RNA in the ALI BEAS-2B cells in vitro was stable at 0.15L/minute but decayed at high flowrates. The ESP device and the resulting model were applied to in vitro studies (i.e., viability and IL-8 expression) of toluene SOA using ALI BEAS-2B cells with a flowrate of 0.15L/minute, and no cellular RNA decay occurred. Copyright © 2017. Published by Elsevier Ltd.

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System@@

EPA Science Inventory

In 2007, the National Research Council envisioned the need for inexpensive, rapid, cell-based toxicity testing methods relevant to human health. in vitro screening approaches have largely addressed these problems by using robotics and automation. However, the challenge is that ma...

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System#

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the cha...

Molluscan cells in culture: primary cell cultures and cell lines

PubMed Central

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

PubMed

Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

2015-07-15

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells

PubMed Central

Li, Nianzhen; Tourovskaia, Anna; Folch, Albert

2013-01-01

The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology—consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium—has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). Micro fabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, the medium composition, as well as the type of neighboring cells surrounding the microenvironment of the cell. In addition, microtechnology is conceptually well suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. Here we review a variety of applications of microfabrication in cell culture studies, with an emphasis on the biology of various cell types. PMID:15139302

[Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

PubMed

Gordeeva, O F; Nikonova, T M; Lifantseva, N V

2009-01-01

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

Development of three-dimensional collagen scaffolds with controlled architecture for cell migration studies using breast cancer cell lines.

PubMed

Campbell, Jonathan J; Husmann, Anke; Hume, Robert D; Watson, Christine J; Cameron, Ruth E

2017-01-01

Cancer is characterized by cell heterogeneity and the development of 3D in vitro assays that can distinguish more invasive or migratory phenotypes could enhance diagnosis or drug discovery. 3D collagen scaffolds have been used to develop analogues of complex tissues in vitro and are suited to routine biochemical and immunological assays. We sought to increase 3D model tractability and modulate the migration rate of seeded cells using an ice-templating technique to create either directional/anisotropic or non-directional/isotropic porous architectures within cross-linked collagen scaffolds. Anisotropic scaffolds supported the enhanced migration of an invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. In addition, MDA-MB-468 showed increased migration upon epithelial-to-mesenchymal transition (EMT) in anisotropic scaffolds. The provision of controlled architecture in this system may act both to increase assay robustness and as a tuneable parameter to capture detection of a migrated population within a set time, with consequences for primary tumour migration analysis. The separation of invasive clones from a cancer biomass with in vitro platforms could enhance drug development and diagnosis testing by contributing assay metrics including migration rate, as well as modelling cell-cell and cell-matrix interaction in a system compatible with routine histopathological testing. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Thicker three-dimensional tissue from a "symbiotic recycling system" combining mammalian cells and algae.

PubMed

Haraguchi, Yuji; Kagawa, Yuki; Sakaguchi, Katsuhisa; Matsuura, Katsuhisa; Shimizu, Tatsuya; Okano, Teruo

2017-01-31

In this paper, we report an in vitro co-culture system that combines mammalian cells and algae, Chlorococcum littorale, to create a three-dimensional (3-D) tissue. While the C2C12 mouse myoblasts and rat cardiac cells consumed oxygen actively, intense oxygen production was accounted for by the algae even in the co-culture system. Although cell metabolism within thicker cardiac cell-layered tissues showed anaerobic respiration, the introduction of innovative co-cultivation partially changed the metabolism to aerobic respiration. Moreover, the amount of glucose consumption and lactate production in the cardiac tissues and the amount of ammonia in the culture media decreased significantly when co-cultivated with algae. In the cardiac tissues devoid of algae, delamination was observed histologically, and the release of creatine kinase (CK) from the tissues showed severe cardiac cell damage. On the other hand, the layered cell tissues with algae were observed to be in a good histological condition, with less than one-fifth decline in CK release. The co-cultivation with algae improved the culture condition of the thicker tissues, resulting in the formation of 160 μm-thick cardiac tissues. Thus, the present study proposes the possibility of creating an in vitro "symbiotic recycling system" composed of mammalian cells and algae.

Implantable wireless powered light emitting diode (LED) for near-infrared photoimmunotherapy: device development and experimental assessment in vitro and in vivo.

PubMed

Nakajima, Kohei; Kimura, Toshihiro; Takakura, Hideo; Yoshikawa, Yasuo; Kameda, Atsushi; Shindo, Takayuki; Sato, Kazuhide; Kobayashi, Hisataka; Ogawa, Mikako

2018-04-13

The aim of this study was to develop and assess a novel implantable, wireless-powered, light-emitting diode (LED) for near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT is a recently developed cancer therapy that uses NIR light and antibody-photosensitizer conjugates and is able to induce cancer-specific cell death. Due to limited light penetration depth it is currently unable to treat tumors in deep tissues. Use of implanted LED might potentially overcome this limitation. The wireless LED system was able to emit NIR light up to a distance of 20 cm from the transmitter coil by using low magnetic fields as compliant with limits for use in humans. Results indicated that the LED system was able to kill tumor cells in vitro and to suppress tumor growth in implanted tumor-bearing mice. Results indicated that the proposed implantable wireless LED system was able to suppress tumor growth in vivo . These results are encouraging as wireless LED systems such as the one here developed might be a possible solution to treat tumors in deep regions in humans. Further research in this area would be important. An implantable LED system was developed. It consisted of a LED capsule including two LED sources and a receiver coil coupled with an external coil and power source. Wireless power transmission was guaranteed by using electromagnetic induction. The system was tested in vitro by using EGFR-expressing cells and HER2-expressing cells. The system was also tested in vivo in tumor-bearing mice.

A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells.

PubMed

Magistri, Marco; Khoury, Nathalie; Mazza, Emilia Maria Cristina; Velmeshev, Dmitry; Lee, Jae K; Bicciato, Silvio; Tsoulfas, Pantelis; Faghihi, Mohammad Ali

2016-11-01

Astrocytes are a morphologically and functionally heterogeneous population of cells that play critical roles in neurodevelopment and in the regulation of central nervous system homeostasis. Studies of human astrocytes have been hampered by the lack of specific molecular markers and by the difficulties associated with purifying and culturing astrocytes from adult human brains. Human neural progenitor cells (NPCs) with self-renewal and multipotent properties represent an appealing model system to gain insight into the developmental genetics and function of human astrocytes, but a comprehensive molecular characterization that confirms the validity of this cellular system is still missing. Here we used an unbiased transcriptomic analysis to characterize in vitro culture of human NPCs and to define the gene expression programs activated during the differentiation of these cells into astrocytes using FBS or the combination of CNTF and BMP4. Our results demonstrate that in vitro cultures of human NPCs isolated during the gliogenic phase of neurodevelopment mainly consist of radial glial cells (RGCs) and glia-restricted progenitor cells. In these cells the combination of CNTF and BMP4 activates the JAK/STAT and SMAD signaling cascades, leading to the inhibition of oligodendrocytes lineage commitment and activation of astrocytes differentiation. On the other hand, FBS-derived astrocytes have properties of reactive astrocytes. Our work suggests that in vitro culture of human NPCs represents a valuable cellular system to study human disorders characterized by impairment of astrocytes development and function. Our datasets represent an important resource for researchers studying human astrocytes development and might set the basis for the discovery of novel human-specific astrocyte markers. © 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

EPA Science Inventory

Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells.

PubMed

Yuri, Shunsuke; Nishikawa, Masaki; Yanagawa, Naomi; Jo, Oak D; Yanagawa, Norimoto

2017-02-14

A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Adenovirus-specific T-cell Subsets in Human Peripheral Blood and After IFN-γ Immunomagnetic Selection.

PubMed

Qian, Chongsheng; Wang, Yingying; Cai, Huili; Laroye, Caroline; De Carvalho Bittencourt, Marcelo; Clement, Laurence; Stoltz, Jean-François; Decot, Véronique; Reppel, Loïc; Bensoussan, Danièle

2016-01-01

Adoptive antiviral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is becoming an alternative treatment for viral infection after hematopoietic stem cell transplantation. The T memory stem cell (TSCM) subset was recently described as exhibiting self-renewal and multipotency properties which are required for sustained efficacy in vivo. We wondered if such a crucial subset for immunotherapy was present in VSTs. We identified, by flow cytometry, TSCM in adenovirus (ADV)-specific interferon (IFN)-γ+ T cells before and after IFN-γ-based immunomagnetic selection, and analyzed the distribution of the main T-cell subsets in VSTs: naive T cells (TN), TSCM, T central memory cells (TCM), T effector memory cell (TEM), and effector T cells (TEFF). In this study all of the different T-cell subsets were observed in the blood sample from healthy donor ADV-VSTs, both before and after IFN-γ-based immunomagnetic selection. As the IFN-γ-based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets, we observed that TEM was always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation and especially after expansion in vitro. Comparing T-cell subpopulation profiles before and after in vitro expansion, we observed that in vitro cell culture with interleukin-2 resulted in a significant expansion of TN-like, TCM, TEM, and TEFF subsets in CD4IFN-γ T cells and of TCM and TEM subsets only in CD8IFN-γ T cells. We demonstrated the presence of all T-cell subsets in IFN-γ VSTs including the TSCM subpopulation, although this was weakly selected by the IFN-γ-based immunomagnetic selection system.

A model-based approach for automated in vitro cell tracking and chemotaxis analyses.

PubMed

Debeir, Olivier; Camby, Isabelle; Kiss, Robert; Van Ham, Philippe; Decaestecker, Christine

2004-07-01

Chemotaxis may be studied in two main ways: 1) counting cells passing through an insert (e.g., using Boyden chambers), and 2) directly observing cell cultures (e.g., using Dunn chambers), both in response to stationary concentration gradients. This article promotes the use of Dunn chambers and in vitro cell-tracking, achieved by video microscopy coupled with automatic image analysis software, in order to extract quantitative and qualitative measurements characterizing the response of cells to a diffusible chemical agent. Previously, we set up a videomicroscopy system coupled with image analysis software that was able to compute cell trajectories from in vitro cell cultures. In the present study, we are introducing a new software increasing the application field of this system to chemotaxis studies. This software is based on an adapted version of the active contour methodology, enabling each cell to be efficiently tracked for hours and resulting in detailed descriptions of individual cell trajectories. The major advantages of this method come from an improved robustness with respect to variability in cell morphologies between different cell lines and dynamical changes in cell shape during cell migration. Moreover, the software includes a very small number of parameters which do not require overly sensitive tuning. Finally, the running time of the software is very short, allowing improved possibilities in acquisition frequency and, consequently, improved descriptions of complex cell trajectories, i.e. trajectories including cell division and cell crossing. We validated this software on several artificial and real cell culture experiments in Dunn chambers also including comparisons with manual (human-controlled) analyses. We developed new software and data analysis tools for automated cell tracking which enable cell chemotaxis to be efficiently analyzed. Copyright 2004 Wiley-Liss, Inc.

In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.

PubMed

Xiang, Jie; Wu, Dai-Chen; Chen, Yuanting; Paulson, Robert F

2015-03-12

Tissue hypoxia induces a systemic response designed to increase oxygen delivery to tissues. One component of this response is increased erythropoiesis. Steady-state erythropoiesis is primarily homeostatic, producing new erythrocytes to replace old erythrocytes removed from circulation by the spleen. In response to anemia, the situation is different. New erythrocytes must be rapidly made to increase hemoglobin levels. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best characterized in the mouse, where it is extramedullary and utilizes progenitors and signals that are distinct from steady-state erythropoiesis. In this report, we use an in vitro culture system that recapitulates the in vivo development of stress erythroid progenitors. We identify cell-surface markers that delineate a series of stress erythroid progenitors with increasing maturity. In addition, we use this in vitro culture system to expand human stress erythroid progenitor cells that express analogous cell-surface markers. Consistent with previous suggestions that human stress erythropoiesis is similar to fetal erythropoiesis, we demonstrate that human stress erythroid progenitors express fetal hemoglobin upon differentiation. These data demonstrate that similar to murine bone marrow, human bone marrow contains cells that can generate BMP4-dependent stress erythroid burst-forming units when cultured under stress erythropoiesis conditions. © 2015 by The American Society of Hematology.

An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

PubMed Central

Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

2016-01-01

The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

Gamma Radiation Induces Micronucleated Reticulocytes in 3-D Bone Marrow Bioreactors in Vitro

PubMed Central

Sun, Hongliang; Dertinger, Stephen D.; Hyrien, Ollivier; David Wu, J. H.; Chen, Yuhchyau

2009-01-01

Radiation injury to the bone marrow is potentially lethal due to the potent DNA-damaging effects on cells of the hematopoietic system, including bone marrow stem cell, progenitor, and the precursor cell populations. Investigation of radiation genotoxic effects on bone marrow progenitor/precursor cells has been challenged by the lack of optimal in vitro surrogate organ culture systems, and the overall difficulty to sustain lineage-specific proliferation and differentiation of hematopoiesis in vitro. We report the investigation of radiation genotoxic effects in bone marrow cultures of C57Bl/6 mice established in 3-D bioreactors, which sustain long-term bone marrow cultures. For these studies, genotoxicity is measured by the induction of micronucleated reticulocytes (MN-RET). The kinetics and dose-response relationship of MN-RET induction in response to gamma-radiation of bioreactor-maintained bone marrow cultures are presented. Our data showed that 3-D long-term bone marrow cultures had sustained erythropoiesis capable of generating reticulocytes up to 8 weeks. The peak time-interval of viable cell output and percentage of reticulocytes increased steadily and reached the initial peak between the 14th to 21st days after inoculations. This was followed by a rebound or staying relatively constant until week 8. The percentage of MN-RET reached the maximum between 24 and 32 hours post 1 Gy gamma-ray. There was a near linear MN-RET induction by gamma radiation from 0 Gy to 1.0 Gy, followed by an attenuated increase to 1.5 – 2.0 Gy. The MN-RET response showed a downtrend beyond 2 Gy. Our data suggest that bone marrow culture in the 3-D bioreactor may be a useful organ culture system for the investigation of radiation genotoxic effect in vitro. PMID:19786117

Immune modulation properties of herbal plant leaves: Phyllanthus niruri aqueous extract on immune cells of tuberculosis patient - in vitro study.

PubMed

Putri, Denise Utami; Rintiswati, Ning; Soesatyo, Marsetyawan Hne; Haryana, Sofia Mubarika

2018-02-01

Disease progression in Tuberculosis (TB) is dependent on host's immune system. Phyllanthus niruri, a traditional herb, has long been used to boost immune system in Indonesian society. This study aimed to observe the potential role of P. niruri in inducing immune cells activity in TB patients by in vitro approach. Peripheral blood mononuclear cells (PBMCs) and macrophages were collected from active pulmonary TB patients. After stimulation with graded doses of P. niruri aqueous extract, cell proliferation, phagocytic activity and nitric oxide (NO) release were analysed. P. niruri aqueous extract induced proliferation of PBMCs, increased NO release, and improved macrophages phagocytic activity. These effects were observed in a dose-dependent manner. This may lead to further research for the potential role of P. niruri as immunomodulatory adjuvant therapy for TB patients.

Perfusion Stirred-Tank Bioreactors for 3D Differentiation of Human Neural Stem Cells.

PubMed

Simão, Daniel; Arez, Francisca; Terasso, Ana P; Pinto, Catarina; Sousa, Marcos F Q; Brito, Catarina; Alves, Paula M

2016-01-01

Therapeutic breakthroughs in neurological disorders have been hampered by the lack of accurate central nervous system (CNS) models. The development of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of new therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmental, anatomic, and physiological) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity, etc.). Recapitulation of CNS phenotypic and functional features in vitro requires the implementation of advanced culture strategies, such as 3D culture systems, which enable to mimic the in vivo structural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. The development of robust and scalable processes for the 3D differentiation of hNSC can improve the accuracy of early stage development in preclinical research. In this context, the use of software-controlled stirred-tank bioreactors (STB) provides an efficient technological platform for hNSC aggregation and differentiation. This system enables to monitor and control important physicochemical parameters for hNSC culture, such as dissolved oxygen. Importantly, the adoption of a perfusion operation mode allows a stable flow of nutrients and differentiation/neurotrophic factors, while clearing the toxic by-products. This contributes to a setting closer to the physiological, by mimicking the in vivo microenvironment. In this chapter, we address the technical requirements and procedures for the implementation of 3D differentiation strategies of hNSC, by operating STB under perfusion mode for long-term cultures. This strategy is suitable for the generation of human 3D neural in vitro models, which can be used to feed high-throughput screening platforms, contributing to expand the available in vitro tools for drug screening and toxicological studies.

In vitro antigen-induced, antigen-specific antibody production in man. Specific and polyclonal components, kinetics, and cellular requirements

PubMed Central

1981-01-01

A highly specific and reproducible antigen-induced, antigen-specific culture and assay system for antibody production by human peripheral blood B lymphocytes has been developed. The system is clearly T cell and monocyte dependent and is independent of exogenous mitogens. The major factors in our ability to trigger specific antibody production with antigen alone have been the use of extremely low concentrations of antigen in vitro (doses several orders of magnitude below those inducing a peak blastogenic response), careful attention to in vitro cell density and culture vessel geometry, and appreciation of the kinetics of the circulating antigen-inducible B cell repertoire. A dichotomy and overlap between antigen-induced, antigen-specific and antigen-induced, polyclonal responses was observed in the study of doubly immunized individuals. Whereas antibody responses highly specific for the antigen in culture were observed under one set of culture conditions (flat-bottomed vessels, 1.5 x 10(6) cells), switching to another culture system (round-bottomed vessels, 5 x 10(5) cells) resulted in polyclonal responses to antigen. Despite these culture condition-related differences in the induction of antibody synthesis, the suppression of specific antibody production that occurred at high concentrations of antigen was specific only for the antigen in culture. The capability to easily and reproducibly look at truly antigen-induced, antigen specific antibody production should be a major tool in furthering the understanding of human B cell activation and immunoregulation. PMID:6169778

In vitro models.

PubMed

Mather, Jennie Powell

2012-02-01

The current resurgence of interest in the cancer stem cell (CSC) hypothesis as possibly providing a unifying theory of cancer biology is fueled by the growing body of work on normal adult tissue stem cells and the promise that CSC may hold the key to one of the central problems of clinical oncology: tumor recurrence. Many studies suggest that the microenvironment plays a role, perhaps a seminal one, in cancer development and progression. In addition, the possibility that the stem cell-like component of tumors is capable of rapid and reversible changes of phenotype raises questions concerning studies with these populations and the application of what we learn to the clinical situation. These types of questions are extremely difficult to study using in vivo models or freshly isolated cells. Established cell lines grown in defined conditions provide important model systems for these studies. There are three types of in vitro models for CSCs: (a) selected subpopulations of existing tumor lines (derived from serum-containing medium; (b) creation of lines from tumor or normal cells by genetic manipulation; or (c) direct in vitro selection of CSC from tumors or sorted tumor cells using defined serum-free conditions. We review the problems associated with creating and maintaining in vitro cultures of CSCs and the progress to date on the establishment of these important models. Copyright © 2011 AlphaMed Press.

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

PubMed

El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

Experimental evidence for killing the resistant cells and raising the efficacy and decreasing the toxicity of cytostatics and irradiation by mixtures of the agents of the passive antitumor defense system in the case of various tumor and normal cell lines in vitro.

PubMed

Kulcsár, Gyula

2009-02-01

Despite the substantial decline of the immune system in AIDS, only a few kinds of tumors increase in incidence. This shows that the immune system has no absolute role in the prevention of tumors. Therefore, the fact that tumors do not develop in the majority of the population during their lifetime indicates the existence of other defense system(s). According to our hypothesis, the defense is made by certain substances of the circulatory system. Earlier, on the basis of this hypothesis, we experimentally selected 16 substances of the circulatory system and demonstrated that the mixture of them (called active mixture) had a cytotoxic effect (inducing apoptosis) in vitro and in vivo on different tumor cell lines, but not on normal cells and animals. In this paper, we provide evidence that different cytostatic drugs or irradiation in combination with the active mixture killed significantly more cancer cells, compared with either treatments alone. The active mixture decreased, to a certain extent, the toxicity of cytostatics and irradiation on normal cells, but the most important result was that the active mixture destroyed the multidrug-resistant cells. Our results provide the possibility to improve the efficacy and reduce the side-effects of chemotherapy and radiation therapy and to prevent the relapse by killing the resistant cells.

Biotechnological approaches to the treatment of aspermatogenic men

PubMed Central

Aponte, Pedro Manuel; Schlatt, Stefan; de Franca, Luiz Renato

2013-01-01

Aspermatogenesis is a severe impairment of spermatogenesis in which germ cells are completely lacking or present in an immature form, which results in sterility in approximately 25% of patients. Because assisted reproduction techniques require mature germ cells, biotechnology is a valuable tool for rescuing fertility while maintaining biological fatherhood. However, this process involves, for instance, the differentiation of preexisting immature germ cells or the production/derivation of sperm from somatic cells. This review critically addresses four potential techniques: sperm derivation in vitro, germ stem cell transplantation, xenologous systems, and haploidization. Sperm derivation in vitro is already feasible in fish and mammals through organ culture or 3D systems, and it is very useful in conditions of germ cell arrest or in type II Sertoli-cell-only syndrome. Patients afflicted by type I Sertoli-cell-only syndrome could also benefit from gamete derivation from induced pluripotent stem cells of somatic origin, and human haploid-like cells have already been obtained by using this novel methodology. In the absence of alternative strategies to generate sperm in vitro, in germ cells transplantation fertility is restored by placing donor cells in the recipient germ-cell-free seminiferous epithelium, which has proven effective in conditions of spermatogonial arrest. Grafting also provides an approach for ex-vivo generation of mature sperm, particularly using prepubertal testis tissue. Although less feasible, haploidization is an option for creating gametes based on biological cloning technology. In conclusion, the aforementioned promising techniques remain largely experimental and still require extensive research, which should address, among other concerns, ethical and biosafety issues, such as gamete epigenetic status, ploidy, and chromatin integrity. PMID:23503966

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

PubMed

Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

2018-04-13

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

Simulation of bone resorption-repair coupling in vitro.

PubMed

Jones, S J; Gray, C; Boyde, A

1994-10-01

In the normal adult human skeleton, new bone formation by osteoblasts restores the contours of bone surfaces following osteoclastic bone resorption, but the evidence for resorption-repair coupling remains circumstantial. To investigate whether sites of prior resorption, more than the surrounding unresorbed surface, attract osteoblasts or stimulate them to proliferate or make new matrix, we developed a simple in vitro system in which resorption-repair coupling occurs. Resorption pits were produced in mammalian dentine or bone slabs by culturing chick bone-derived cells on them for 2-3 days. The chick cells were swept off and the substrata reseeded with rat calvarial osteoblastic cells, which make bone nodules in vitro, for periods of up to 8 weeks. Cell positions and new bone formation were investigated by ordinary light microscopy, fluorescence and reflection confocal laser microscopy, and SEM, in stained and unstained samples. There was no evidence that the osteoblasts were especially attracted to, or influenced by, the sites of resorption in dentine or bone before cell confluence was reached. Bone formation was identified by light microscopy by the accumulation of matrix, staining with alizarin and calcein and by von Kossa's method, and confirmed by scanning electron microscopy (SEM) by using backscattered electron (BSE) and transmitted electron imaging of unembedded samples and BSE imaging of micro-milled embedded material. These new bone patches were located initially in the resorption pits. The model in vitro system may throw new light on the factors that control resorption-repair coupling in the mineralised tissues in vivo.

Endocrine disrupters--testing strategies to assess human hazard.

PubMed

Baker, V A

2001-01-01

During the last decade an hypothesis has been developed linking certain chemicals (natural and synthetic) to observed and suspected adverse effects on reproduction in both wildlife and humans. The issue of 'endocrine disruption' originally focused on chemicals that mimic the action of the natural hormone oestrogen. However, the concern is now encompassing effects on the whole endocrine system. In response to public awareness, regulatory agencies (including the US EPA) and the OECD are formulating potential testing strategies and have begun the process of validating defined tests to systematically assess chemicals for their endocrine-disrupting activities. In order to investigate chemicals that have the potential to cause endocrine disruption, a large number of in vitro and in vivo assays have been identified. In vitro test systems (particularly when used in combination) offer the possibility of providing an early screen for large numbers of chemicals and can be useful in characterising the mechanism of action and potency. In vitro assays in widespread use for the screening/characterisation of endocrine disrupting potential include hormone receptor ligand binding assays (determination of the ability of a chemical to bind to the hormone receptor), cell proliferation assays (analysis of the ability of a chemical to stimulate growth of oestrogen sensitive cells), reporter gene assays in yeast or mammalian cells (analysis of the ability of a chemical to stimulate the transcription of a reporter gene construct in cell culture), and the analysis of the regulation of endogenous oestrogen sensitive genes in cell lines. However, in vitro assays do not always reliably predict the outcome in vivo due to differences in metabolic capabilities of the test systems used and the diverse range of mechanisms by which endocrine disrupting chemicals may act. Therefore a complementary battery of short- and long-term in vitro and in vivo assays (that assess both receptor and non-receptor mediated mechanisms of action) seems the most appropriate way at present of assessing the potential endocrine disrupting activities of chemicals. At Unilever we have used a combination of in vitro assays (receptor binding, reporter gene and cell proliferation assays) together with short-term in vivo tests (uterotrophic assay in immature rodents) to examine the oestrogenic potential of a large number of chemicals. An evaluation of the advantages and limitations of these methods is provided. Finally, any potential test system needs to be validated and standardized before the information generated can be for the identification of hazard, and possibly for risk assessment purposes.

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

Manipulation of immune system via immortal bone marrow stem cells.

PubMed

Ruedl, Christiane; Khameneh, Hanif Javanmard; Karjalainen, Klaus

2008-09-01

Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells engineered to over-express NUP98-HOXB4 fusion protein support self-renewal of physiologically normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to differentiate in vivo or in vitro to any hematopoietic lineage.

In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.

PubMed

Nayak, Bibhukalyan Prasad; Goh, James Cho Hong; Toh, Siew Lok; Satpathy, Gyan Ranjan

2010-03-01

Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro. The current study has two objectives. The first objective is to demonstrate functional coculture of bone marrow-derived stem cells (BMSCs) with mature bone cells/ligament fibroblasts as evidenced by gap-junctional communication in vitro. The second objective is to investigate the role of BMSCs in the regeneration of fibrocartilage within the coculture. Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control). Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control. The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration. The results open up a path to envisage a composite graft preinstalled with enthesial fibrocartilage using a stem cell-based coculture system.

COMPARISON OF THE ESTROGENIC POTENCIES OF ESTRADIOL, ETHYNYLESTRADIOL, DIETHYLSTILBESTROL, NONYLPHENOL AND METHOXYCHLOR IN VIVO AND IN VITRO

EPA Science Inventory

Five natural, pharmaceutical, or xenobiotic chemicals (17b-estradiol, ethynylestradiol, diethystilbestrol, nonylphenol, methoxychlor) were tested in two in vitro (MCF-7 breast tumor cell proliferation [E-screen], yeast estrogen system [YES]), and one in vivo (male sheepshead min...

Untangling cell tracks: Quantifying cell migration by time lapse image data analysis.

PubMed

Svensson, Carl-Magnus; Medyukhina, Anna; Belyaev, Ivan; Al-Zaben, Naim; Figge, Marc Thilo

2018-03-01

Automated microscopy has given researchers access to great amounts of live cell imaging data from in vitro and in vivo experiments. Much focus has been put on extracting cell tracks from such data using a plethora of segmentation and tracking algorithms, but further analysis is normally required to draw biologically relevant conclusions. Such relevant conclusions may be whether the migration is directed or not, whether the population has homogeneous or heterogeneous migration patterns. This review focuses on the analysis of cell migration data that are extracted from time lapse images. We discuss a range of measures and models used to analyze cell tracks independent of the biological system or the way the tracks were obtained. For single-cell migration, we focus on measures and models giving examples of biological systems where they have been applied, for example, migration of bacteria, fibroblasts, and immune cells. For collective migration, we describe the model systems wound healing, neural crest migration, and Drosophila gastrulation and discuss methods for cell migration within these systems. We also discuss the role of the extracellular matrix and subsequent differences between track analysis in vitro and in vivo. Besides methods and measures, we are putting special focus on the need for openly available data and code, as well as a lack of common vocabulary in cell track analysis. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Inhibition of intra-Golgi transport in vitro by mitotic kinase.

PubMed

Stuart, R A; Mackay, D; Adamczewski, J; Warren, G

1993-02-25

It has previously been shown that exocytic and endocytic membrane traffic are inhibited in mitotic mammalian cells. Here we have used a cell-free intra-Golgi transport assay supplemented with heterologous cytosols to mimic this effect in vitro. Cytosols with high histone kinase activity, made either from mitotic cells or by cyclin A treatment of interphase cells, inhibited intra-Golgi transport by up to 75%. Inhibition of transport was reversed by the kinase inhibitor staurosporine or by reduction in ATP levels leading to inactivation of histone kinase. The data indicate that cell cycle control of intra-Golgi transport is due to a reversible modification of cytosol, and this assay system may be used to study the molecular mechanism of mitotic transport inhibition in mammalian cells.

Apoptosis induced by cold shock in vitro is dependent on cell growth phase.

PubMed

Soloff, B L; Nagle, W A; Moss, A J; Henle, K J; Crawford, J T

1987-06-15

Chinese hamster V79 fibroblast cells were exposed to brief periods of cold but non-freezing temperatures at different points on the population growth curve. Upon rewarming, cells at the transition from logarithmic to stationary growth exhibited apoptosis (programmed cell death). Cells in other stages of growth, or after reentry into logarithmic growth by refeeding, did not exhibit apoptosis. Apoptosis was expressed by marked cytoplasmic blebbing, by a characteristic non-random fragmentation of DNA into nucleosomal-sized pieces, and by loss of colony-forming ability. The data suggest that cold shock served as a stimulus for susceptible cells to undergo apoptosis. Thus, the experiments describe a new in vitro system for studying the mechanisms of apoptosis.

Pharmacodynamics of cisplatin in human head and neck cancer: correlation between platinum content, DNA adduct levels and drug sensitivity in vitro and in vivo

PubMed Central

Welters, M J P; Fichtinger-Schepman, A M J; Baan, R A; Jacobs-Bergmans, A J; Kegel, A; van der Vijgh, W J F; Braakhuis, B J M

1999-01-01

Total platinum contents and cisplatin–DNA adduct levels were determined in vivo in xenografted tumour tissues in mice and in vitro in cultured tumour cells of head and neck squamous cell carcinoma (HNSCC), and correlated with sensitivity to cisplatin. In vivo, a panel of five HNSCC tumour lines growing as xenografts in nude mice was used. In vitro, the panel consisted of five HNSCC cell lines, of which four had an in vivo equivalent. Sensitivity to cisplatin varied three- to sevenfold among cell lines and tumours respectively. However, the ranking of the sensitivities of the tumour lines (in vivo), also after reinjection of the cultured tumour cells, did not coincide with that of the corresponding cell lines, which showed that cell culture systems are not representative for the in vivo situation. Both in vitro and in vivo, however, significant correlations were found between total platinum levels, measured by atomic absorption spectrophotometry (AAS), and tumour response to cisplatin therapy at all time points tested. The levels of the two major cisplatin–DNA adduct types were determined by a recently developed and improved32P post-labelling assay at various time points after cisplatin treatment. Evidence is presented that the platinum–AG adduct, in which platinum is bound to guanine and an adjacent adenine, may be the cytotoxic lesion because a significant correlation was found between the platinum–AG levels and the sensitivities in our panel of HNSCC, in vitro as well as in vivo. This correlation with the platinum–AG levels was established at 1 h (in vitro) and 3 h (in vivo) after the start of the cisplatin treatment, which emphasizes the importance of early sampling. © 1999 Cancer Research Campaign PMID:10408697

Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

PubMed Central

Wang, Jun; Wu, Chengxiong; Hu, Ning; Zhou, Jie; Du, Liping; Wang, Ping

2012-01-01

Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA), the electric cell-substrate impedance sensing (ECIS) technique, and the light addressable potentiometric sensor (LAPS). The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology. PMID:25585708

An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

PubMed Central

Janebodin, Kajohnkiart; Buranaphatthana, Worakanya; Ieronimakis, Nicholas; Hays, Aislinn L.; Reyes, Morayma

2013-01-01

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors. PMID:23841093

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

PubMed

Mandenius, Carl-Fredrik; Andersson, Tommy B; Alves, Paula M; Batzl-Hartmann, Christine; Björquist, Petter; Carrondo, Manuel J T; Chesne, Christophe; Coecke, Sandra; Edsbagge, Josefina; Fredriksson, J Magnus; Gerlach, Jörg C; Heinzle, Elmar; Ingelman-Sundberg, Magnus; Johansson, Inger; Küppers-Munther, Barbara; Müller-Vieira, Ursula; Noor, Fozia; Zeilinger, Katrin

2011-05-01

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance. 2011 FRAME.

Brain-Derived Neurotrophic Factor Induces Cell Survival and the Migration of Murine Adult Hippocampal Precursor Cells During Differentiation In Vitro.

PubMed

Ortiz-López, Leonardo; Vega-Rivera, Nelly Maritza; Babu, Harish; Ramírez-Rodríguez, Gerardo Bernabé

2017-01-01

The generation of new neurons during adulthood involves local precursor cell migration and terminal differentiation in the dentate gyrus. These events are influenced by the hippocampal microenvironment. Brain-derived neurotrophic factor (BDNF) is relevant for hippocampal neuronal development and behavior. Interestingly, studies that have been performed in controlled in vitro systems that involve isolated precursor cells that were derived from the dentate gyrus (AHPCs) have shown that BDNF induces the activation of the TrkB receptor and, consequentially, might activate signaling pathways that favor survival and neuronal differentiation. Based on the fact that the cellular events of AHPCs that are induced by single factors can be studied in this controlled in vitro system, we investigated the ability of BDNF and the involvement of protein kinase C (PKC), as one of the TrkB-downstream activated signaling proteins, in the regulation of migration, here reflected by motility, of AHPCs. Precursor cells were cultured following a concentration-response curve (1-640 ng/ml) for 24 or 96 h. We found that BDNF favored cell survival without altering the viability under culture proliferative conditions of the AHPCs. Concomitantly, glial- and neuronal-differentiated precursor cells increased as a consequence of survival promoted by BDNF. Additionally, pharmacological approaches showed that BDNF (40 ng/ml)-induced migration of AHPCs was blocked with the compounds K252a and GF109203x, which prevent the activation of TrkB and PKC, respectively. The results indicate that in the in vitro migration of differentiated AHPCs it is involved the BDNF and TrkB cascade. Our results provide additional information about the mechanism by which BDNF impacts adult neurogenesis in the hippocampus.

In vivo UVA irradiation of mouse is more efficient in promoting pulmonary melanoma metastasis than in vitro

PubMed Central

2011-01-01

Background We have previously shown in vitro that UVA increases the adhesiveness of mouse B16-F1 melanoma cells to endothelium. We have also shown in vivo that UVA exposure of C57BL/6 mice, i.v. injected with B16-F1 cells, increases formation of pulmonary colonies of melanoma. The aim of the present animal study was to confirm the previously observed in vivo UVA effect and to determine whether in vitro UVA-exposure of melanoma cells, prior the i.v. injection, will have an enhancing effect on the pulmonary colonization capacity of melanoma cells. As a second aim, UVA-derived immunosuppression was determined. Methods Mice were i.v. injected with B16-F1 cells into the tail vein and then immediately exposed to UVA. Alternatively, to study the effect of UVA-induced adhesiveness on the colonization capacity of B16-F1 melanoma, cells were in vitro exposed prior to i.v. injection. Fourteen days after injection, lungs were collected and the number of pulmonary nodules was determined under dissecting microscope. The UVA-derived immunosuppression was measured by standard contact hypersensitivity assay. Results and Discussion Obtained results have confirmed that mice, i.v. injected with B16-F1 cells and thereafter exposed to UVA, developed 4-times more of melanoma colonies in lungs as compared with the UVA non-exposed group (p < 0.01). The in vitro exposure of melanoma cells prior to their injection into mice, led only to induction of 1.5-times more of pulmonary tumor nodules, being however a statistically non-significant change. The obtained results postulate that the UVA-induced changes in the adhesive properties of melanoma cells do not alone account for the 4-fold increase in the pulmonary tumor formation. Instead, it suggests that some systemic effect in a mouse might be responsible for the increased metastasis formation. Indeed, UVA was found to induce moderate systemic immunosuppression, which effect might contribute to the UVA-induced melanoma metastasis in mice lungs. PMID:21645404

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

PubMed Central

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells. PMID:26882313

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture.

PubMed

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

PubMed

Thiery, J P; Boyer, B; Tucker, G; Gavrilovic, J; Valles, A M

1988-01-01

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal pattern, is involved in tissue formation and in key processes of tumour spread.

Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

EPA Science Inventory

When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

Pentacle gold-copper alloy nanocrystals: a new system for entering male germ cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Lin, Yu; He, Rong; Sun, Liping; Yang, Yushan; Li, Wenqing; Sun, Fei

2016-12-01

Gold-based nanocrystals have attracted considerable attention for drug delivery and biological applications due to their distinct shapes. However, overcoming biological barriers is a hard and inevitable problem, which restricts medical applications of nanomaterials in vivo. Seeking for an efficient transportation to penetrate biological barriers is a common need. There are three barriers: blood-testis barrier, blood-placenta barrier, and blood-brain barrier. Here, we pay close attention to the blood-testis barrier. We found that the pentacle gold-copper alloy nanocrystals not only could enter GC-2 cells in vitro in a short time, but also could overcome the blood-testis barrier and enter male germ cells in vivo. Furthermore, we demonstrated that the entrance efficiency would become much higher in the development stages. The results also suggested that the pentacle gold-copper alloy nanocrystals could easier enter to germ cells in the pathological condition. This system could be a new method for theranostics in the reproductive system.

Characterisation of the cancer-associated glucocorticoid system: key role of 11β-hydroxysteroid dehydrogenase type 2.

PubMed

Cirillo, Nicola; Morgan, David J; Pedicillo, Maria Carmela; Celentano, Antonio; Lo Muzio, Lorenzo; McCullough, Michael J; Prime, Stephen S

2017-09-26

Recent studies have shown that production of cortisol not only takes place in several non-adrenal peripheral tissues such as epithelial cells but, also, the local inter-conversion between cortisone and cortisol is regulated by the 11β-hydroxysteroid dehydrogenases (11β-HSDs). However, little is known about the activity of this non-adrenal glucocorticoid system in cancers. The presence of a functioning glucocorticoid system was assessed in human skin squamous cell carcinoma (SCC) and melanoma and further, in 16 epithelial cell lines from 8 different tissue types using ELISA, western blotting and immunofluorescence. 11β-HSD2 was inhibited both pharmacologically and by siRNA technology. Naïve CD8 + T cells were used to test the paracrine effects of cancer-derived cortisol on the immune system in vitro. Functional assays included cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical data of 11β-HSD expression were generated using tissue microarrays of 40 cases of human SCCs as well as a database featuring 315 cancer cases from 15 different tissues. We show that cortisol production is a common feature of malignant cells and has paracrine functions. Cortisol production correlated with the magnitude of glucocorticoid receptor (GR)-dependent inhibition of tumour-specific CD8 + T cells in vitro. 11β-HSDs were detectable in human skin SCCs and melanoma. Analyses of publicly available protein expression data of 11β-HSDs demonstrated that 11β-HSD1 and -HSD2 were dysregulated in the majority (73%) of malignancies. Pharmacological manipulation of 11β-HSD2 activity by 18β-glycyrrhetinic acid (GA) and silencing by specific siRNAs modulated the bioavailability of cortisol. Cortisol also acted in an autocrine manner and promoted cell invasion in vitro and cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical analyses using tissue microarrays showed that expression of 11β-HSD2 was significantly reduced in human SCCs of the skin. The results demonstrate evidence of a cancer-associated glucocorticoid system and show for the first time, the functional significance of cancer-derived cortisol in tumour progression.

Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.

PubMed

Knittel, T; Aurisch, S; Neubauer, K; Eichhorst, S; Ramadori, G

1996-08-01

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.

Sonic hedgehog protein promotes proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.

PubMed

Warzecha, Jörg; Göttig, Stephan; Brüning, Christian; Lindhorst, Elmar; Arabmothlagh, Mohammad; Kurth, Andreas

2006-10-01

Sonic hedgehog (Shh) protein is known to be an important signaling protein in early embryonic development. Also, Shh is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb and in the spine. The impact of Shh on adult stem cells, human bone marrow-derived mesenchymal stem cells (MSCs), was tested. The MSCs were treated either with recombinant Sonic hedgehog protein (r-Shh) or with transforming growth factor-beta 1 (TGF-beta(1)) as a positive control in vitro for 3 weeks. The effects on cartilaginous differentiation and proliferation were assayed. MSCs when treated with either Shh or TGF-beta(1) showed expression of cartilage markers aggrecan, Sox9, CEP-68, and collagen type II and X within 3 weeks. Only r-Shh-treated cells showed a very strong cell proliferation and much higher BrdU incorporation in cell assay systems. These are the first data that indicate an important role of Shh for the induction of cartilage production by MSCs in vitro.

Effect of methylmercury on the rat mast cell degranulation

NASA Astrophysics Data System (ADS)

Graevskaya, E. E.; Yasutake, A.; Aramai, R.; Rubin, A. B.

2003-05-01

Methylmercury is the well-known neurotoxicant as weil as a modulator of the immune system. We investigated the effects of MeHg on the rat mast cell degranulation induced by nonimmunological stimuli (the selective liberator of histamine, compound 48/80, and calcium ionophore A23187) both in vivo and in vitro. In 8, 12 and 15 days afterthe final administration of MeHg we observed the suppression of calcium ionophore A23187-and 48/80-induced histamine release, which enhanced with time. In experiments in vitro incubation of peritoneal mast cells with MeHg alone in the dose range 10^{-8} to 10^{-6} did not induce mast cell degranulation, however modified the activation of mast cells by compound 48/80, and calcium ionophore A23187. We observed activation of stimulated secretion by preliminary incubation with low dose of MeHg 10^{-8} M and inhibition by dose of MeHg 10^{-6} M. These results show that MeHg treatment can modify mast cell function in vivo and in vitro and provide insight into the understanding what role this cell has in the pathogenesis of Minamata disease-comlected disorders.

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

PubMed Central

Ohta, Naoki; Kato, Yasuhiko; Watanabe, Hajime; Mori, Hirotada; Matsuura, Tomoaki

2016-01-01

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach. PMID:27808179

Toxicity Screening of Volatile Chemicals Using a Novel Air-Liquid Interface In Vitro Exposure System

EPA Science Inventory

Traditional in vitro dosing methods require, for example, the addition of particulate matter (PM), PM extracts, or chemicals in dimethyl sulfoxide (DMSO) or water into cell culture medium. However, about 10% of chemicals nominated for study in the U.S Environmental Protection Age...

ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE OUTGROWTH, NEURONAL POLARIZATION AND SYNAPTOGENESIS IN RAT CORTICAL NEURONS USING HIGH CONTENT IMAGE ANALYSIS

EPA Science Inventory

There is a need for efficient, cost-effective methods for screening and prioritization of potential developmental neurotoxicants. One approach uses in vitro cell culture models that can recapitulate the critical processes of nervous system development. In vitro, primary cultures ...

The data of establishing a three-dimensional culture system for in vitro recapitulation and mechanism exploration of tumor satellite formation during cancer cell transition.

PubMed

Chen, Chun-Nan; Chen, You-Tzung; Yang, Tsung-Lin

2017-12-01

Tumor satellite formation is an indicator of cancer invasiveness and correlates with recurrence, metastasis, and poorer prognosis. By analyzing pathological specimens, tumor satellites formed at the tumor-host interface reflect the phenomena of epithelial-mesenchymal transition. It is impossible to reveal the dynamic processes and the decisive factors of tumor satellite formation using clinicopathological approaches alone. Therefore, establishment of an in vitro system to monitor the phenomena is important to explicitly elucidate underlying mechanisms. In this study, we explored the feasibility of creating an in vitro three-dimensional collagen culture system to recapitulate the process of tumor satellite formation. This data presented here are referred to the research article (Chen et al., 2017) [1]. Using this model, the dynamic process of tumor satellite formation could be recapitulated in different types of human cancer cells. Induced by calcium deprivation, the treated cells increased the incidence and migratory distance of tumor satellites. E-cadherin internalization and invadopodia formation were enhanced by calcium deprivation and were associated with cellular dynamic change during tumor satellite formation. The data confirmed the utility of this culture system to recapitulate dynamic cellular alteration and to explore the potential mechanisms of tumor satellite formation.

Sofosbuvir inhibits hepatitis A virus replication in vitro assessed by a cell-based fluorescent reporter system.

PubMed

Jiang, Wang; Muhammad, Fawad; Ma, Pengjuan; Liu, Xiyu; Long, Gang

2018-06-01

Hepatitis A virus (HAV) infection remains a major cause of acute hepatitis worldwide and even leads to fulminant hepatitis. For screening antivirals against HAV in vitro, we develop a cell-based fluorescent reporter system named Huh-7.5.1-GA, in which HAV infection is visualized by green fluorescence protein (GFP) translocation from the cytosol into the nucleus. The reliability of Huh-7.5.1-GA for antiviral studies is validated by IFN-α, a known inhibitor of HAV replication, which impedes GFP translocation. Utilizing this in-vitro reporter system, we find that sofosbuvir, an FDA approved prodrug for the treatment of chronic hepatitis C, disturbs GFP translocation and inhibits HAV replication efficiently. In addition, we find that inhibition of HAV by sofosbuvir is hepatic-cell dependent, with IC50 (half-maximal inhibitory concentration) being 6.3 μM and 9.9 μM in Huh-7.5.1, quantified separately by RT-qPCR and image-based analysis. Therefore, our reporter system may serve as a high-throughput platform for screening potent antivirals against HAV. Sofosbuvir may be considered for treatment of hepatitis A, especially in re-infected patients who undergo liver transplantation due to HAV-induced liver failure. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of Nanoparticles on Complement System in Cell Culture Model

DTIC Science & Technology

2006-09-15

case complement activation considerably differs between nanoparticles , being the highest in case of fullerene, ferric oxide and aluminium oxide ... oxide (CdO; 1 µm), manganese oxide (MnO2; 1-2 µm), and tungsten (W; 27 µm) were assessed. Additionally the effects of nanoparticles coated with...using in vitro system. Obtained results indicate that: 1. Nanoparticles toxicity in vitro can’t be measured using methods which were designed

BK virus has tropism for human salivary gland cells in vitro: Implications for transmission

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

Jeffers, Liesl K.; Madden, Vicki; Webster-Cyriaque, Jennifer, E-mail: jennifer@med.unc.ed

Background: In this study, it was determined that BKV is shed in saliva and an in vitro model system was developed whereby BKV can productively infect both submandibular (HSG) and parotid (HSY) salivary gland cell lines. Results: BKV was detected in oral fluids using quantitative real-time PCR (QRTPCR). BKV infection was determined using quantitative RT-PCR, immunofluorescence and immunoblotting assays. The infectivity of BKV was inhibited by pre-incubation of the virus with gangliosides that saturated the major capsid protein, VP1, halting receptor mediated BKV entry into salivary gland cells. Examination of infected cultures by transmission electron microscopy revealed 45-50 nm BKmore » virions clearly visible within the cells. Subsequent to infection, encapsidated BK virus was detected in the supernatant. Conclusion: We thus demonstrated that BKV was detected in oral fluids and that BK infection and replication occur in vitro in salivary gland cells. These data collectively suggest the potential for BKV oral route of transmission and oral pathogenesis.« less

A fluid–structure interaction model to characterize bone cell stimulation in parallel-plate flow chamber systems

PubMed Central

Vaughan, T. J.; Haugh, M. G.; McNamara, L. M.

2013-01-01

Bone continuously adapts its internal structure to accommodate the functional demands of its mechanical environment and strain-induced flow of interstitial fluid is believed to be the primary mediator of mechanical stimuli to bone cells in vivo. In vitro investigations have shown that bone cells produce important biochemical signals in response to fluid flow applied using parallel-plate flow chamber (PPFC) systems. However, the exact mechanical stimulus experienced by the cells within these systems remains unclear. To fully understand this behaviour represents a most challenging multi-physics problem involving the interaction between deformable cellular structures and adjacent fluid flows. In this study, we use a fluid–structure interaction computational approach to investigate the nature of the mechanical stimulus being applied to a single osteoblast cell under fluid flow within a PPFC system. The analysis decouples the contribution of pressure and shear stress on cellular deformation and for the first time highlights that cell strain under flow is dominated by the pressure in the PPFC system rather than the applied shear stress. Furthermore, it was found that strains imparted on the cell membrane were relatively low whereas significant strain amplification occurred at the cell–substrate interface. These results suggest that strain transfer through focal attachments at the base of the cell are the primary mediators of mechanical signals to the cell under flow in a PPFC system. Such information is vital in order to correctly interpret biological responses of bone cells under in vitro stimulation and elucidate the mechanisms associated with mechanotransduction in vivo. PMID:23365189

A mathematical model of antibody-dependent cellular cytotoxicity (ADCC).

PubMed

Hoffman, F; Gavaghan, D; Osborne, J; Barrett, I P; You, T; Ghadially, H; Sainson, R; Wilkinson, R W; Byrne, H M

2018-01-07

Immunotherapies exploit the immune system to target and kill cancer cells, while sparing healthy tissue. Antibody therapies, an important class of immunotherapies, involve the binding to specific antigens on the surface of the tumour cells of antibodies that activate natural killer (NK) cells to kill the tumour cells. Preclinical assessment of molecules that may cause antibody-dependent cellular cytotoxicity (ADCC) involves co-culturing cancer cells, NK cells and antibody in vitro for several hours and measuring subsequent levels of tumour cell lysis. Here we develop a mathematical model of such an in vitro ADCC assay, formulated as a system of time-dependent ordinary differential equations and in which NK cells kill cancer cells at a rate which depends on the amount of antibody bound to each cancer cell. Numerical simulations generated using experimentally-based parameter estimates reveal that the system evolves on two timescales: a fast timescale on which antibodies bind to receptors on the surface of the tumour cells, and NK cells form complexes with the cancer cells, and a longer time-scale on which the NK cells kill the cancer cells. We construct approximate model solutions on each timescale, and show that they are in good agreement with numerical simulations of the full system. Our results show how the processes involved in ADCC change as the initial concentration of antibody and NK-cancer cell ratio are varied. We use these results to explain what information about the tumour cell kill rate can be extracted from the cytotoxicity assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro.

PubMed

van Vollenstee, Fiona A; Jackson, Carlo; Hoffmann, Danie; Potgieter, Marnie; Durandt, Chrisna; Pepper, Michael S

2016-10-01

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

Computational Modeling and Simulation of Developmental ...

EPA Pesticide Factsheets

Standard practice for assessing developmental toxicity is the observation of apical endpoints (intrauterine death, fetal growth retardation, structural malformations) in pregnant rats/rabbits following exposure during organogenesis. EPA’s computational toxicology research program (ToxCast) generated vast in vitro cellular and molecular effects data on >1858 chemicals in >600 high-throughput screening (HTS) assays. The diversity of assays has been increased for developmental toxicity with several HTS platforms, including the devTOX-quickPredict assay from Stemina Biomarker Discovery utilizing the human embryonic stem cell line (H9). Translating these HTS data into higher order-predictions of developmental toxicity is a significant challenge. Here, we address the application of computational systems models that recapitulate the kinematics of dynamical cell signaling networks (e.g., SHH, FGF, BMP, retinoids) in a CompuCell3D.org modeling environment. Examples include angiogenesis (angiodysplasia) and dysmorphogenesis. Being numerically responsive to perturbation, these models are amenable to data integration for systems Toxicology and Adverse Outcome Pathways (AOPs). The AOP simulation outputs predict potential phenotypes based on the in vitro HTS data ToxCast. A heuristic computational intelligence framework that recapitulates the kinematics of dynamical cell signaling networks in the embryo, together with the in vitro profiling data, produce quantitative predic

Development of an in vitro PIG-A gene mutation assay in human cells

PubMed Central

Rees, Benjamin J; Tate, Matthew; Lynch, Anthony M; Thornton, Catherine A; Jenkins, Gareth J; Walmsley, Richard M; Johnson, George E

2017-01-01

Abstract Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella ‘Ames’ reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell’s extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing. PMID:28057708

Microphysiological systems meet hiPSC technology - New tools for disease modeling of liver infections in basic research and drug development.

PubMed

Raasch, Martin; Fritsche, Enrico; Kurtz, Andreas; Bauer, Michael; Mosig, Alexander S

2018-06-14

Complex cell culture models such as microphysiological models (MPS) mimicking human liver functionality in vitro are in the spotlight as alternative to conventional cell culture and animal models. Promising techniques like microfluidic cell culture or micropatterning by 3D bioprinting are gaining increasing importance for the development of MPS to address the needs for more predictivity and cost efficiency. In this context, human induced pluripotent stem cells (hiPSCs) offer new perspectives for the development of advanced liver-on-chip systems by recreating an in vivo like microenvironment that supports the reliable differentiation of hiPSCs to hepatocyte-like cells (HLC). In this review we will summarize current protocols of HLC generation and highlight recently established MPS suitable to resemble physiological hepatocyte function in vitro. In addition, we are discussing potential applications of liver MPS for disease modeling related to systemic or direct liver infections and the use of MPS in testing of new drug candidates. Copyright © 2018. Published by Elsevier B.V.

In vitro protein expression: an emerging alternative to cell-based approaches.

PubMed

He, Mingyue

2011-04-30

Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

Modeling human disease using organotypic cultures.

PubMed

Schweiger, Pawel J; Jensen, Kim B

2016-12-01

Reliable disease models are needed in order to improve quality of healthcare. This includes gaining better understanding of disease mechanisms, developing new therapeutic interventions and personalizing treatment. Up-to-date, the majority of our knowledge about disease states comes from in vivo animal models and in vitro cell culture systems. However, it has been exceedingly difficult to model disease at the tissue level. Since recently, the gap between cell line studies and in vivo modeling has been narrowing thanks to progress in biomaterials and stem cell research. Development of reliable 3D culture systems has enabled a rapid expansion of sophisticated in vitro models. Here we focus on some of the latest advances and future perspectives in 3D organoids for human disease modeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Case Study: Organotypic human in vitro models of embryonic morphogenetic fusion

EPA Science Inventory

Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell...

Insights into female germ cell biology: from in vivo development to in vitro derivations.

PubMed

Jung, Dajung; Kee, Kehkooi

2015-01-01

Understanding the mechanisms of human germ cell biology is important for developing infertility treatments. However, little is known about the mechanisms that regulate human gametogenesis due to the difficulties in collecting samples, especially germ cells during fetal development. In contrast to the mitotic arrest of spermatogonia stem cells in the fetal testis, female germ cells proceed into meiosis and began folliculogenesis in fetal ovaries. Regulations of these developmental events, including the initiation of meiosis and the endowment of primordial follicles, remain an enigma. Studying the molecular mechanisms of female germ cell biology in the human ovary has been mostly limited to spatiotemporal characterizations of genes or proteins. Recent efforts in utilizing in vitro differentiation system of stem cells to derive germ cells have allowed researchers to begin studying molecular mechanisms during human germ cell development. Meanwhile, the possibility of isolating female germline stem cells in adult ovaries also excites researchers and generates many debates. This review will mainly focus on presenting and discussing recent in vivo and in vitro studies on female germ cell biology in human. The topics will highlight the progress made in understanding the three main stages of germ cell developments: namely, primordial germ cell formation, meiotic initiation, and folliculogenesis.

Three-dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells.

PubMed

Zhan, Kang; Lin, Miao; Liu, MingMei; Sui, YangNan; Babekir, Haitham Mohammed; Zhao, GuoQi

2017-05-01

Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro. © 2016 Japanese Society of Animal Science.

Metabolite formation kinetics and intrinsic clearance of phenacetin, tolbutamide, alprazolam, and midazolam in adenoviral cytochrome P450-transfected HepG2 cells and comparison with hepatocytes and in vivo.

PubMed

Donato, M Teresa; Hallifax, David; Picazo, Laura; Castell, José V; Houston, J Brian; Gomez-Lechón, M José; Lahoz, Agustin

2010-09-01

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the K(m) or S(50) values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CL(int) (or CL(max)), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CL(int) values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1'-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CL(int) for the substrates studied (in a range of 20-200% in vivo observed CL(int)). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CL(int)) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.

An integrated dispersion preparation, characterization and in vitro dosimetry methodology for engineered nanomaterials

PubMed Central

DeLoid, Glen M.; Cohen, Joel M.; Pyrgiotakis, Georgios; Demokritou, Philip

2018-01-01

Summary Evidence continues to grow of the importance of in vitro and in vivo dosimetry in the hazard assessment and ranking of engineered nanomaterials (ENMs). Accurate dose metrics are particularly important for in vitro cellular screening to assess the potential health risks or bioactivity of ENMs. In order to ensure meaningful and reproducible quantification of in vitro dose, with consistent measurement and reporting between laboratories, it is necessary to adopt standardized and integrated methodologies for 1) generation of stable ENM suspensions in cell culture media, 2) colloidal characterization of suspended ENMs, particularly properties that determine particle kinetics in an in vitro system (size distribution and formed agglomerate effective density), and 3) robust numerical fate and transport modeling for accurate determination of ENM dose delivered to cells over the course of the in vitro exposure. Here we present such an integrated comprehensive protocol based on such a methodology for in vitro dosimetry, including detailed standardized procedures for each of these three critical steps. The entire protocol requires approximately 6-12 hours to complete. PMID:28102836

The selective effect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells

NASA Astrophysics Data System (ADS)

Duan, J.; Lu, X.; He, G.

2017-01-01

In this work, a co-culture system with liver cancer cell line HepG2 and normal cell line L02 is used to investigate the selective effect on cancer and normal cells by plasma activated medium (PAM), which is closer to the real environment where cancer cells develop. Besides, the co-culture system is a better model to study the selective effect than the widely used separate culture systems, where the cancer cell line and normal cell line are cultured independently. By using the co-culture system, it is found that there is an optimum dose of PAM to induce significant cancer cell apoptosis while keeping minimum damage to normal cells.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip.

PubMed

Dauth, Stephanie; Maoz, Ben M; Sheehy, Sean P; Hemphill, Matthew A; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M; Budnik, Bogdan; Parker, Kevin Kit

2017-03-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. Copyright © 2017 the American Physiological Society.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip

PubMed Central

Dauth, Stephanie; Maoz, Ben M.; Sheehy, Sean P.; Hemphill, Matthew A.; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M.; Budnik, Bogdan

2017-01-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. PMID:28031399

[The role of the serotonin system in the stress response of various cells

NASA Technical Reports Server (NTRS)

Belzhelarskaia, S. N.; Satton, F. F.; Sutton, F. (Principal Investigator)

2003-01-01

The recombinant mouse brain serotonin receptor (5HT1c) was used to study the response of plant cells and oocytes to a stress signal activated by the serotonin-serotonin receptor interaction and associated Ca2+ flow. Based on plant expression vectors, recombinant constructs were obtained to direct production of 5HT1c fused with the green fluorescent protein in plant cells. The mRNAs for hybrid proteins were synthesized in an in vitro transcription system. The expression and function of the hybrid protein and the function of the associated ion channels were electrophysiologically studied in Xenopus laevis oocytes injected with the hybrid mRNA. The hybrid protein was functional and changed the operation of the Ca2+ channel in oocytes. To study the expression of the hybrid constructs in plant cells, the in vitro transcription product was inoculated in tobacco leaves, which then fluoresced.

Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

PubMed

Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

2018-01-01

Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

Isolation and in vitro binding of mating type plus fertilization tubules from Chlamydomonas.

PubMed

Wilson, Nedra F

2008-01-01

During fertilization in Chlamydomonas, adhesion and fusion of gametes occur at the tip of specialized regions of the plasma membrane, known as mating structures. The mating type minus (mt[-]) structure is a slightly raised dome-shaped region located at the apical end of the cell body. In contrast, the activated mating type plus (mt[+]) structure is an actin-filled, microvillouslike organelle. Interestingly, a similar type of "fusion organelle" is conserved across diverse groups. Chlamydomonas provides an ideal model system for studying the process of gametic cell fusion in that it is amenable to genetic manipulations as well as cell and molecular biological approaches. Moreover, the ease of culturing Chlamydomonas combined with the ability to isolate the mt(+) fertilization tubule and the development of in vitro assays for adhesion makes it an ideal system for biochemical studies focused on dissecting the molecular mechanisms that underlie the complex process of gametic cell fusion.

The cell biology of Drosophila wing metamorphosis in vitro.

PubMed

Milner, Martin J; Muir, Jonathan

1987-03-01

We have examined the metamorphosis of the wing imaginal disc of Drosophila during culture in vitro in the continuous presence of 20-hydroxy ecdysone (0.1 μg/ ml). We find that the sequence of cellular changes in the wing blade during culture closely match those occurring in situ, involving two periods at which the dorsal and ventral surfaces are joined only by cell processes containing trans-alar microtubule arrays. Good pupal and imaginal cuticle secretion is found in this system.

Cytotoxic lymphocytes in Hashimoto thyroiditis: an in vitro assay system using 51Cr-labelled chicken red blood cells coated with thyroglobulin

PubMed Central

Calder, Elizabeth A.; Penhale, W. J.; Barnes, E. W.; Irvine, W. J.

1973-01-01

An in vitro method is described to detect lymphocytes in patients with Hashimoto thyroiditis that are cytotoxic to thyroglobulin-coated chicken red blood cells. Using this technique, the cytotoxic index of lymphocytes from patients with Hashimoto thyroiditis was 25·46±3·81 (SEM), which is significantly different from that obtained with lymphocytes from control subjects, 6·28±0·80. PMID:4740396

Carbon Ion Irradiated Neural Injury Induced the Peripheral Immune Effects in Vitro or in Vivo

PubMed Central

Lei, Runhong; Zhao, Tuo; Li, Qiang; Wang, Xiao; Ma, Hong; Deng, Yulin

2015-01-01

Carbon ion radiation is a promising treatment for brain cancer; however, the immune system involved long-term systemic effects evoke a concern of complementary and alternative therapies in clinical treatment. To clarify radiotherapy caused fundamental changes in peripheral immune system, examinations were performed based on established models in vitro and in vivo. We found that brain-localized carbon ion radiation of neural cells induced complex changes in the peripheral blood, thymus, and spleen at one, two, and three months after its application. Atrophy, apoptosis, and abnormal T-cell distributions were observed in rats receiving a single high dose of radiation. Radiation downregulated the expression of proteins involved in T-cell development at the transcriptional level and increased the proportion of CD3+CD4−CD8+ T-cells in the thymus and the proportion of CD3+CD4+CD8− T-cells in the spleen. These data show that brain irradiation severely affects the peripheral immune system, even at relatively long times after irradiation. In addition, they provide valuable information that will implement the design of biological-based strategies that will aid brain cancer patients suffering from the long-term side effects of radiation. PMID:26633364

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

NASA Technical Reports Server (NTRS)

Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

2003-01-01

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Peptide-functionalized quantum dots for potential applications in the imaging and treatment of obesity.

PubMed

Thovhogi, Ntevheleni; Sibuyi, Nicole Remaliah Samantha; Onani, Martin Opiyo; Meyer, Mervin; Madiehe, Abram Madimabe

2018-01-01

Obesity is a worldwide epidemic affecting millions of people. The current pharmacological treatment of obesity remains limited and ineffective due to drugs' undesirable side effects. Hence, there is a need for novel or improved strategies for long-term therapies that will help prevent the disease progression into other chronic diseases. Nanotechnology holds the future for the treatment of obesity because of its versatility, as shown by improved drug efficiency and safety in cancer clinical trials. Nano-based drug delivery systems could potentially do the same for obesity through targeted drug delivery. This study investigated the use of peptide-functionalized quantum dots (QDs) for the imaging of prohibitin (PHB)-expressing cells in vitro and in diet-induced obese rats, which could potentially be used as nanocarriers of antiobesity drugs. Cadmium (Cd)-based QDs were functionalized with an adipose homing peptide (AHP) and injected intravenously into lean and obese Wistar rats. Biodistribution of the QDs was analyzed by an IVIS ® Lumina XR imaging system and inductively coupled plasma optical emission spectroscopy (ICP-OES). For in vitro studies, PHB-expressing (Caco-2 and MCF-7) and non-PHB-expressing (KMST-6 and CHO) cells were exposed to either unfunctionalized QDs (QD625) or AHP-functionalized QDs (AHP-QD625) and analyzed by fluorescence microscopy. AHP-QD625 accumulated significantly in PHB-expressing cells in vitro when compared with non-PHB-expressing cells. In vivo data indicated that QD625 accumulated mainly in the reticuloendothelial system (RES) organs, while the AHP-QD625 accumulated mostly in the white adipose tissues (WATs). AHP-functionalized QDs were successfully and selectively delivered to the PHB-expressing cells in vitro (Caco-2 and MCF-7 cells) and in the WAT vasculature in vivo. This nanotechnology-based approach could potentially be used for dual targeted drug delivery and molecular imaging of adipose tissues in obese patients in real time.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions

DOE PAGES

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.; ...

2015-07-30

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Enzymatic reactions in confined environments

NASA Astrophysics Data System (ADS)

Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

2016-05-01

Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

New experimental models of the blood-brain barrier for CNS drug discovery

PubMed Central

Kaisar, Mohammad A.; Sajja, Ravi K.; Prasad, Shikha; Abhyankar, Vinay V.; Liles, Taylor; Cucullo, Luca

2017-01-01

Introduction The blood-brain barrier (BBB) is a dynamic biological interface which actively controls the passage of substances between the blood and the central nervous system (CNS). From a biological and functional standpoint, the BBB plays a crucial role in maintaining brain homeostasis inasmuch that deterioration of BBB functions are prodromal to many CNS disorders. Conversely, the BBB hinders the delivery of drugs targeting the brain to treat a variety of neurological diseases. Area covered This article reviews recent technological improvements and innovation in the field of BBB modeling including static and dynamic cell-based platforms, microfluidic systems and the use of stem cells and 3D printing technologies. Additionally, the authors laid out a roadmap for the integration of microfluidics and stem cell biology as a holistic approach for the development of novel in vitro BBB platforms. Expert opinion Development of effective CNS drugs has been hindered by the lack of reliable strategies to mimic the BBB and cerebrovascular impairments in vitro. Technological advancements in BBB modeling have fostered the development of highly integrative and quasi- physiological in vitro platforms to support the process of drug discovery. These advanced in vitro tools are likely to further current understanding of the cerebrovascular modulatory mechanisms. PMID:27782770

Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity.

PubMed

Elferink, M G L; Olinga, P; Draaisma, A L; Merema, M T; Bauerschmidt, S; Polman, J; Schoonen, W G; Groothuis, G M M

2008-06-15

The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such as Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl(4), fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.

Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity

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

Elferink, M.G.L.; Olinga, P.; Draaisma, A.L.

2008-06-15

The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such asmore » Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl{sub 4}, fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.« less

Development of Acyclovir-Loaded Albumin Nanoparticles and Improvement of Acyclovir Permeation Across Human Corneal Epithelial T Cells.

PubMed

Suwannoi, Panita; Chomnawang, Mullika; Sarisuta, Narong; Reichl, Stephan; Müller-Goymann, Christel C

2017-12-01

The aim of the present study was to develop acyclovir (ACV) ocular drug delivery systems of bovine serum albumin (BSA) nanoparticles as well as to assess their in vitro transcorneal permeation across human corneal epithelial (HCE-T) cell multilayers. The ACV-loaded BSA nanoparticles were prepared by desolvation method along with physicochemical characterization, cytotoxicity, as well as in vitro transcorneal permeation studies across HCE-T cell multilayers. The nanoparticles appeared to be spherical in shape and nearly uniform in size of about 200 nm. The size of nanoparticles became smaller with decreasing BSA concentration, while the ratios of water to ethanol seemed not to affect the size. Increasing the amount of ethanol in desolvation process led to significant reduction of drug entrapment of nanoparticles with smaller size and more uniformity. The ACV-loaded BSA nanoparticles prepared were shown to have no cytotoxic effect on HCE-T cells used in permeation studies. The in vitro transcorneal permeation results revealed that ACV could permeate through the HCE-T cell multilayers significantly higher from BSA nanoparticles than from aqueous ACV solutions. The ACV-loaded BSA nanoparticles could be prepared by desolvation method without glutaraldehyde in the formulation. ACV could increasingly permeate through the multilayers of HCE-T cells from the ACV-loaded BSA nanoparticles. Therefore, the ACV-loaded BSA nanoparticles could be a highly potential ocular drug delivery system.

Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions.

PubMed

Loret, Thomas; Peyret, Emmanuel; Dubreuil, Marielle; Aguerre-Chariol, Olivier; Bressot, Christophe; le Bihan, Olivier; Amodeo, Tanguy; Trouiller, Bénédicte; Braun, Anne; Egles, Christophe; Lacroix, Ghislaine

2016-11-03

Recently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO 2 and one nano-CeO 2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the air-liquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed. Deposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used. We showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.

High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system.

PubMed

Martin, G A; Kawaguchi, R; Lam, Y; DeGiovanni, A; Fukushima, M; Mutter, W

2001-10-01

The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

3D printed nervous system on a chip.

PubMed

Johnson, Blake N; Lancaster, Karen Z; Hogue, Ian B; Meng, Fanben; Kong, Yong Lin; Enquist, Lynn W; McAlpine, Michael C

2016-04-21

Bioinspired organ-level in vitro platforms are emerging as effective technologies for fundamental research, drug discovery, and personalized healthcare. In particular, models for nervous system research are especially important, due to the complexity of neurological phenomena and challenges associated with developing targeted treatment of neurological disorders. Here we introduce an additive manufacturing-based approach in the form of a bioinspired, customizable 3D printed nervous system on a chip (3DNSC) for the study of viral infection in the nervous system. Micro-extrusion 3D printing strategies enabled the assembly of biomimetic scaffold components (microchannels and compartmented chambers) for the alignment of axonal networks and spatial organization of cellular components. Physiologically relevant studies of nervous system infection using the multiscale biomimetic device demonstrated the functionality of the in vitro platform. We found that Schwann cells participate in axon-to-cell viral spread but appear refractory to infection, exhibiting a multiplicity of infection (MOI) of 1.4 genomes per cell. These results suggest that 3D printing is a valuable approach for the prototyping of a customized model nervous system on a chip technology.

Quantitative Analysis of Dendritic Cell Haptotaxis.

PubMed

Schwarz, Jan; Sixt, Michael

2016-01-01

Chemokines are the main guidance cues directing leukocyte migration. Opposed to early assumptions, chemokines do not necessarily act as soluble cues but are often immobilized within tissues, e.g., dendritic cell migration toward lymphatic vessels is guided by a haptotactic gradient of the chemokine CCL21. Controlled assay systems to quantitatively study haptotaxis in vitro are still missing. In this chapter, we describe an in vitro haptotaxis assay optimized for the unique properties of dendritic cells. The chemokine CCL21 is immobilized in a bioactive state, using laser-assisted protein adsorption by photobleaching. The cells follow this immobilized CCL21 gradient in a haptotaxis chamber, which provides three dimensionally confined migration conditions. © 2016 Elsevier Inc. All rights reserved.

In vitro adherence of radioactively labeled Escherichia coli in normal and cystitis-prone females

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

Parsons, C.L.; Anwar, H.; Stauffer, C.

Numerous investigators report data obtained using an in vitro quantitative assay for measuring bacterial adherence to epithelial cells. In the modified assay described here, we eliminated the need for visual counting of bacteria by incorporating the use of radioactively labeled Escherichia coli. This allowed quantitation of bacterial adherence to as many as 50,000 vaginal cells, whereas the visual counting system limits the determination to perhaps 50 cells. Using the modified method, we found no statistically significant differences among values for adherence of E. coli type 04 to the vaginal cells of control and cystitis-prone women at either pH 6.4 ormore » 4.0.« less

Encapsulated human hepatocellular carcinoma cells by alginate gel beads as an in vitro metastasis model

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

Xu, Xiao-xi; Liu, Chang; University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049

2013-08-15

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and often forms metastases, which are the most important prognostic factors. For further elucidation of the mechanism underlying the progression and metastasis of HCC, a culture system mimicking the in vivo tumor microenvironment is needed. In this study, we investigated the metastatic ability of HCC cells cultured within alginate gel (ALG) beads. In the culture system, HCC cells formed spheroids by proliferation and maintained in nuclear abnormalities. The gene and protein expression of metastasis-related molecules was increased in ALG beads, compared with the traditional adhesion culture. Furthermore, several gene expressionmore » levels in ALG bead culture system were even closer to liver cancer tissues. More importantly, in vitro invasion assay showed that the invasion cells derived from ALG beads was 7.8-fold higher than adhesion cells. Our results indicated that the in vitro three-dimensional (3D) model based on ALG beads increased metastatic ability compared with adhesion culture, even partly mimicked the in vivo tumor tissues. Moreover, due to the controllable preparation conditions, steady characteristics and production at large-scale, the 3D ALG bead model would become an important tool used in the high-throughput screening of anti-metastasis drugs and the metastatic mechanism research. -- Highlights: •We established a 3D metastasis model mimicking the metastatic ability in vivo. •The invasion ability of cells derived from our model was increased significantly. •The model is easy to reproduce, convenient to handle, and amenable for large-scale.« less

E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.

PubMed

Park, Kyeong-Su; Kim, Ju Hee; Shin, Hee Won; Chung, Kyung-Sook; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

2015-10-26

Missense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method. The stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticancer effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay. Three VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model. These data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL.

A 3-D Cardiac Muscle Construct for Exploring Adult Marrow Stem Cell Based Myocardial Regeneration

PubMed Central

Valarmathi, Mani T.; Goodwin, Richard L.; Fuseler, John W.; Davis, Jeffrey M.; Yost, Michael J.; Potts, Jay D.

2010-01-01

Adult bone marrow stromal cells (BMSCs) are capable of differentiating into cardiomyocyte-like cells in vitro and contribute to myocardial regeneration in vivo. Consequently, BMSCs may potentially play a vital role in cardiac repair and regeneration. However, this concept has been limited by inadequate and inconsistent differentiation of BMSCs into cardiomyocytes along with poor survival and integration of neo-cardiomyocytes after implantation into ischemic myocardium. In order to overcome these barriers and to explore adult stem cell based myocardial regeneration, we have developed an in vitro model of three-dimensional (3-D) cardiac muscle using rat ventricular embryonic cardiomyocytes (ECMs) and BMSCs. When ECMs and BMSCs were seeded sequentially onto a 3-D tubular scaffold engineered from topographically aligned type I collagen fibers and cultured in basal medium for 7, 14, 21, or 28 days, the maturation and co-differentiation into a cardiomyocyte lineage was observed. Phenotypic induction was characterized at morphological, immunological, biochemical and molecular levels. The observed expression of transcripts coding for cardiomyocyte phenotypic markers and the immunolocalization of cardiomyogenic lineage-associated proteins revealed typical expression patterns of neo-cardiomyogenesis. At the biochemical level differentiating cells exhibited appropriate metabolic activity and at the ultrastructural level myofibrillar and sarcomeric organization were indicative of an immature phenotype. Our 3-D co-culture system sustains the ECMs in vitro continuum of differentiation process and simultaneously induces the maturation and differentiation of BMSCs into cardiomyocyte-like cells. Thus, this novel 3-D co-culture system provides a useful in vitro model to investigate the functional role and interplay of developing ECMs and BMSCs during cardiomyogenic differentiation. PMID:20129663

Inhibition of T cell-mediated functions by MVM(i), a parvovirus closely related to minute virus of mice.

PubMed

Engers, H D; Louis, J A; Zubler, R H; Hirt, B

1981-12-01

A purified preparation of MVM(i), a murine parvovirus closely related to minute virus of mice (MVM), was found to inhibit various functions mediated by murine T cells in vitro. Addition of MVM(i) virus to secondary allogeneic mixed leukocyte cultures resulted in the inhibition of both lymphocyte proliferation (3H-thymidine incorporation) and the generation of cytolytic T lymphocyte activity but not interferon production. MVM(i) virus also inhibited the growth and cytolytic activity of several cloned, long-term Lyt-2+ cytolytic T cell lines. Furthermore, the antigen-induced proliferative responses of parasite- (Leishmania) specific Lyt-1+ T cells in vitro was abrogated by the addition of MVM(i) virus to the culture. Finally, the suppression of an in vitro antibody response to SRBC by MVM(i) virus was the result of the inhibition of T helper cells required for the B cell response. These suppressive effects were specific for MVM(i); parallel studies in which the prototype MVM parvovirus was used showed no significant inhibition in the various systems tested.

A Functional High-Throughput Assay of Myelination in Vitro

DTIC Science & Technology

2014-07-01

iPS cells derived from human astrocytes. These cell lines will serve as an excellent source of human cells from which our model systems may be...image the 3D rat dorsal root ganglion ( DRG ) cultures with sufficiently low background as to detect electrically-evoked depolarization events, as...stimulation and recording system specifically for this purpose. Further, we found that the limitations inherent in optimizing speed and FOV may

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.

Systemic administration of thrombin peptide TP508 enhances VEGF-stimulated angiogenesis and attenuates effects of chronic hypoxia

PubMed Central

Olszewska-Pazdrak, Barbara; Carney, Darrell H.

2015-01-01

Revascularization of chronic wounds and ischemic tissue is attenuated by endothelial dysfunction and the inability of angiogenic factors to stimulate angiogenesis. We recently showed that TP508, a nonproteolytic thrombin peptide, increases perfusion and NO-dependent vasodilation in hearts with chronic ischemia and stimulates NO production by endothelial cells. In this study, we investigated systemic in vivo effects of TP508 on VEGF-stimulated angiogenesis in vitro using aortic explants in normoxic and hypoxic conditions. Mice were injected with saline or TP508 and 24h later aortas were removed and cultured to quantify endothelial sprouting. TP508 injection increased endothelial sprouting and potentiated the in vitro response to VEGF. Exposure of control explants to hypoxia inhibited basal and VEGF-stimulated endothelial cell sprouting. This effect of hypoxia was significantly prevented by TP508 injection. Thus, TP508 systemic administration increases responsiveness of aortic endothelial cells to VEGF and diminishes the effect of chronic hypoxia on endothelial cell sprouting. Studies using human endothelial cells in culture suggest that protective effects of TP508 during hypoxia may involve stimulation of endothelial cell NO production. These data suggest potential clinical benefit of using a combination of systemic TP508 and local VEGF as a therapy for revascularization of ischemic tissue. PMID:23594718

Programmable In Vitro Coencapsidation of Guest Proteins for Intracellular Delivery by Virus-like Particles.

PubMed

Dashti, Noor H; Abidin, Rufika S; Sainsbury, Frank

2018-05-22

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages are being developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of bionanotechnology platforms that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of murine polyomavirus (MPyV) that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer, we demonstrate the flexibility in this system to support coencapsidation of multiple proteins. Complementing these ensemble measurements with single-particle analysis by super-resolution microscopy shows that the stochastic nature of coencapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable coencapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

Fail-Safe System against Potential Tumorigenicity after Transplantation of iPSC Derivatives.

PubMed

Itakura, Go; Kawabata, Soya; Ando, Miki; Nishiyama, Yuichiro; Sugai, Keiko; Ozaki, Masahiro; Iida, Tsuyoshi; Ookubo, Toshiki; Kojima, Kota; Kashiwagi, Rei; Yasutake, Kaori; Nakauchi, Hiromitsu; Miyoshi, Hiroyuki; Nagoshi, Narihito; Kohyama, Jun; Iwanami, Akio; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

2017-03-14

Human induced pluripotent stem cells (iPSCs) are promising in regenerative medicine. However, the risks of teratoma formation and the overgrowth of the transplanted cells continue to be major hurdles that must be overcome. Here, we examined the efficacy of the inducible caspase-9 (iCaspase9) gene as a fail-safe against undesired tumorigenic transformation of iPSC-derived somatic cells. We used a lentiviral vector to transduce iCaspase9 into two iPSC lines and assessed its efficacy in vitro and in vivo. In vitro, the iCaspase9 system induced apoptosis in approximately 95% of both iPSCs and iPSC-derived neural stem/progenitor cells (iPSC-NS/PCs). To determine in vivo function, we transplanted iPSC-NS/PCs into the injured spinal cord of NOD/SCID mice. All transplanted cells whose mass effect was hindering motor function recovery were ablated upon transduction of iCaspase9. Our results suggest that the iCaspase9 system may serve as an important countermeasure against post-transplantation adverse events in stem cell transplant therapies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

PubMed

Stoelzle, Sonja; Haythornthwaite, Alison; Kettenhofen, Ralf; Kolossov, Eugen; Bohlen, Heribert; George, Michael; Brüggemann, Andrea; Fertig, Niels

2011-09-01

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

In Vitro Microfluidic Models for Neurodegenerative Disorders.

PubMed

Osaki, Tatsuya; Shin, Yoojin; Sivathanu, Vivek; Campisi, Marco; Kamm, Roger D

2018-01-01

Microfluidic devices enable novel means of emulating neurodegenerative disease pathophysiology in vitro. These organ-on-a-chip systems can potentially reduce animal testing and substitute (or augment) simple 2D culture systems. Reconstituting critical features of neurodegenerative diseases in a biomimetic system using microfluidics can thereby accelerate drug discovery and improve our understanding of the mechanisms of several currently incurable diseases. This review describes latest advances in modeling neurodegenerative diseases in the central nervous system and the peripheral nervous system. First, this study summarizes fundamental advantages of microfluidic devices in the creation of compartmentalized cell culture microenvironments for the co-culture of neurons, glial cells, endothelial cells, and skeletal muscle cells and in their recapitulation of spatiotemporal chemical gradients and mechanical microenvironments. Then, this reviews neurodegenerative-disease-on-a-chip models focusing on Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Finally, this study discusses about current drawbacks of these models and strategies that may overcome them. These organ-on-chip technologies can be useful to be the first line of testing line in drug development and toxicology studies, which can contribute significantly to minimize the phase of animal testing steps. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Culturing primary mouse pancreatic ductal cells.

PubMed

Reichert, Maximilian; Rhim, Andrew D; Rustgi, Anil K

2015-06-01

The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles ductal cells morphologically. To study pancreatic ductal cell (PDC) and pancreatic intraepithelial neoplasia (PanIN)/PDAC biology, it is essential to have reliable in vitro culture conditions. Here we describe a methodology to isolate, culture, and passage PDCs and duct-like cells from the mouse pancreas. It can be used to isolate cells from genetically engineered mouse models (GEMMs), providing a valuable tool to study disease models in vitro to complement in vivo findings. The culture conditions allow epithelial cells to outgrow fibroblast and other "contaminating" cell types within a few passages. However, the resulting cultures, although mostly epithelial, are not completely devoid of fibroblasts. Regardless, this protocol provides guidelines for a robust in vitro culture system to isolate, maintain, and expand primary pancreatic ductal epithelial cells. It can be applied to virtually all GEMMs of pancreatic disease and other diseases and cancers that arise from ductal structures. Because most carcinomas resemble ductal structures, this protocol has utility in the study of other cancers in addition to PDAC, such as breast and prostate cancers. © 2015 Cold Spring Harbor Laboratory Press.

An estimation of the frequency of precursor cells which generate cytotoxic lymphocytes

PubMed Central

1976-01-01

The cell-mediated immune response has been generated in vitro with a polyacrylamide culture system which allows the segregation of foci (clones?) of cytotoxic lymphocytes. Using the method of limiting dilutions, the frequency of precursor cells in CBA spleen cells able to generate a cytotoxic response against DBA mastocytoma is estimated at 1 per 1,700 cells. PMID:1083894

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

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

Katano, Takahito; Ootani, Akifumi; Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501

2013-03-22

Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system withinmore » the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.« less

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

PubMed

Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

2018-05-01

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

Using in vitro models for expression profiling studies on ethanol and drugs of abuse.

PubMed

Thibault, Christelle; Hassan, Sajida; Miles, Michael

2005-03-01

The use of expression profiling with microarrays offers great potential for studying the mechanisms of action of drugs of abuse. Studies with the intact nervous system seem likely to be most relevant to understanding the mechanisms of drug abuse-related behaviours. However, the use of expression profiling with in vitro culture models offers significant advantages for identifying details of cellular signalling actions and toxicity for drugs of abuse. This study discusses general issues of the use of microarrays and cell culture models for studies on drugs of abuse. Specific results from existing studies are also discussed, providing clear examples of relevance for in vitro studies on ethanol, nicotine, opiates, cannabinoids and hallucinogens such as LSD. In addition to providing details on signalling mechanisms relevant to the neurobiology of drugs of abuse, microarray studies on a variety of cell culture systems have also provided important information on mechanisms of cellular/organ toxicity with drugs of abuse. Efforts to integrate genomic studies on drugs of abuse with both in vivo and in vitro models offer the potential for novel mechanistic rigor and physiological relevance.

In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model

PubMed Central

Majety, Meher; Pradel, Leon P.; Gies, Manuela; Ries, Carola H.

2015-01-01

In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME. PMID:26053043

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell-cell adhesion of colorectal cancer cells.

PubMed

Aarons, Cary B; Bajenova, Olga; Andrews, Charles; Heydrick, Stanley; Bushell, Kristen N; Reed, Karen L; Thomas, Peter; Becker, James M; Stucchi, Arthur F

2007-01-01

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

PubMed Central

Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

2014-01-01

STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by mislocalization of actin filament barbed end capping and bundling protein Eps8, and branched actin polymerization protein Arp3. Besides impeding actin dynamics, endocytic vesicle-mediated trafficking and the proper localization of actin regulatory proteins c-Src and annexin II in Sertoli cells were also affected. Results of statistical analysis demonstrate that these findings were not obtained by chance. LIMITATIONS, REASONS FOR CAUTION (i) This study was done in vitro and might not extrapolate to the in vivo state, (ii) conclusions are based on the use of Sertoli cell samples from three men and (iii) it is uncertain if the concentrations of toxicants used in the experiments are reached in vivo. WIDER IMPLICATIONS OF THE FINDINGS Human Sertoli cells cultured in vitro provide a robust model to monitor environmental toxicant-mediated disruption of Sertoli cell BTB function and to study the mechanism(s) of toxicant-induced testicular dysfunction. PMID:24532171

Anticonvulsant hypersensitivity syndrome. In vitro assessment of risk.

PubMed Central

Shear, N H; Spielberg, S P

1988-01-01

Arene oxide metabolites of aromatic anticonvulsants (phenytoin, phenobarbital, and carbamazepine) may be involved in the pathogenesis of hypersensitivity reactions. We investigated 53 patients with clinical sensitivity to anticonvulsants by exposing their lymphocytes in vitro to drug metabolites generated by a murine hepatic microsomal system. The diagnosis of a hypersensitivity reaction was corroborated by in vitro rechallenge for each drug (phenytoin, n = 34; phenobarbital, n = 22; carbamazepine, n = 25) when cytotoxicity (% dead cells) exceeded 3 SD above the mean result for controls. Cross-reactivity among the drugs was noted. 7 out of 10 patients who had received all three anticonvulsants had adverse reactions to each. 40 out of 50 patients tested to all three drugs in vitro were positive to each. Adverse reactions were indistinguishable among anti-convulsants. Skin rash (87%), fever (94%), hepatitis (51%), and hematologic abnormalities (51%) were common clinical features of each drug. 62% of reactions involved more than two organs. Cells from patients' parents exhibited in vitro toxicity that was intermediate between values for controls and patients. In vitro testing can help diagnose hypersensitivity to anticonvulsants. Cells from patients may also be used for prospective individualization of therapy to decrease risk of adverse reaction. Cross-reactivity among the major anticonvulsants is common and should be considered before deciding on alternative therapy. Images PMID:3198757

Preparation of high bioactivity multilayered bone-marrow mesenchymal stem cell sheets for myocardial infarction using a 3D-dynamic system.

PubMed

Wang, Yingwei; Zhang, Jianhua; Qin, Zixi; Fan, Zepei; Lu, Cheng; Chen, Baoxin; Zhao, Jupeng; Li, Xiaojuan; Xiao, Fei; Lin, Xi; Wu, Zheng

2018-05-01

Cell sheet techniques offer a promising future for myocardial infarction (MI) therapy; however, insufficient nutrition supply remains the major limitation in maintaining stem cell bioactivity in vitro. In order to enhance cell sheet mechanical strength and bioactivity, a decellularized porcine pericardium (DPP) scaffold was prepared by the phospholipase A2 method, and aspartic acid was used as a spacer arm to improve the vascular endothelial growth factor crosslink efficiency on the DPP scaffold. Based on this scaffold, multilayered bone marrow mesenchymal stem cell sheets were rapidly constructed, using RAD16-I peptide hydrogel as a temporary 3D scaffold, and cell sheets were cultured in either the 3D-dynamic system (DCcs) or the traditional static condition (SCcs). The multilayered structure, stem cell bioactivity, and ultrastructure of DCcs and SCcs were assessed. The DCcs exhibited lower apoptosis, lower differentiation, and an improved paracrine effect after a 48 h culture in vitro compared to the SCcs. Four groups were set to evaluate the cell sheet effect in rat MI model: sham group, MI control group, DCcs group, and SCcs group. The DCcs group improved cardiac function and decreased the infarcted area compared to the MI control group, while no significant improvements were observed in the SCcs group. Improved cell survival, angiogenesis, and Sca-1 + cell and c-kit + cell amounts were observed in the DCcs group. In conclusion, the DCcs maintained higher stem cell bioactivity by using the 3D-dynamic system to provide sufficient nutrition, and transplanting DCcs significantly improved the cardiac function and angiogenesis. This study provides an efficient method to prepare vascular endothelial growth factor covalent decellularized pericardium scaffold with aspartic acid, and a multilayered bone marrow mesenchymal stem cell (BMSC) sheet is constructed on it using a 3D-dynamic system. The dynamic nutrition supply showed a significant benefit on BMSC bioactivity in vitro, including decreasing cell apoptosis, reducing stem cell differentiation, and improving growth factor secretion. These favorable bioactivity improved BMSC survival, angiogenesis, and cardiac function of the infarcted myocardium. The study highlights the importance of dynamic nutrition supply on maintaining stem cell bioactivity within cell sheet, and it stresses the necessity and significance of setting a standard for assessing cell sheet products before transplantation in the future application. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Anti-allergic activity of 2,4,6-trihydroxy-3-geranylacetophenone (tHGA) via attenuation of IgE-mediated mast cell activation and inhibition of passive systemic anaphylaxis

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

Tan, Ji Wei; Israf, Daud Ahmad; Harith, Hanis Haze

tHGA, a geranyl acetophenone compound originally isolated from a local shrub called Melicope ptelefolia, has been previously reported to prevent ovalbumin-induced allergic airway inflammation in a murine model of allergic asthma by targeting cysteinyl leukotriene synthesis. Mast cells are immune effector cells involved in the pathogenesis of allergic diseases including asthma by releasing cysteinyl leukotrienes. The anti-asthmatic properties of tHGA could be attributed to its inhibitory effect on mast cell degranulation. As mast cell degranulation is an important event in allergic responses, this study aimed to investigate the anti-allergic effects of tHGA in cellular and animal models of IgE-mediated mastmore » cell degranulation. For in vitro model of IgE-mediated mast cell degranulation, DNP-IgE-sensitized RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA to induce degranulation. For IgE-mediated passive systemic anaphylaxis, Sprague Dawley rats were sensitized by intraperitoneal injection of DNP-IgE before challenged with DNP-BSA. Both in vitro and in vivo models showed that tHGA significantly inhibited the release of preformed mediators (β-hexosaminidase and histamine) as well as de novo mediators (interleukin-4, tumour necrosis factor-α, prostaglandin D{sub 2} and leukotriene C{sub 4}). Pre-treatment of tHGA also prevented IgE-challenged RBL-2H3 cells and peritoneal mast cells from undergoing morphological changes associated with mast cell degranulation. These findings indicate that tHGA possesses potent anti-allergic activity via attenuation of IgE-mediated mast cell degranulation and inhibition of IgE-mediated passive systemic anaphylaxis. Thus, tHGA may have the potential to be developed as a mast cell stabilizer for the treatment of allergic diseases in the future. - Highlights: • The in vitro and in vivo mast cell stabilizing effects of tHGA were examined. • tHGA counteracts the plasma membrane deformation in degranulating mast cells. • tHGA attenuates preformed and de novo mediators released by degranulating mast cells. • tHGA prevents in vivo mast cell activation and passive systemic anaphylaxis in rats. • tHGA could be a potential mast cell stabilizer for the treatment of allergic diseases.« less

In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells.

PubMed

Jung, Kwang Bo; Lee, Hana; Son, Ye Seul; Lee, Ji Hye; Cho, Hyun-Soo; Lee, Mi-Ok; Oh, Jung-Hwa; Lee, Jaemin; Kim, Seokho; Jung, Cho-Rok; Kim, Janghwan; Son, Mi-Young

2018-01-01

Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine-specific gene promoters Krüppel-like factor 5 monomeric Cherry (KLF5 mCherry ) and intestine-specific homeobox enhanced green fluorescence protein (ISX eGFP ). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry- or eGFP-expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell-based therapies and preclinical screenings in the intestinal field.-Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.-S., Lee, M.-O., Oh, J.-H., Lee, J., Kim, S., Jung, C.-R., Kim, J., Son, M.-Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. © FASEB.

Solid freeform fabrication and in-vitro response of osteoblast cells of mPEG-PCL-mPEG bone scaffolds.

PubMed

Jiang, Cho-Pei; Chen, Yo-Yu; Hsieh, Ming-Fa; Lee, Hung-Maan

2013-04-01

Bone tissue engineering is an emerging approach to provide viable substitutes for bone regeneration. Poly(ethylene glycol) (PEG) is a good candidate of bone scaffold because of several advantages such as hydrophilicity, biocompatibility, and intrinsic resistance to protein adsorption and cell adhesion. However, its low compressive strength limits application for bone regeneration. Poly(ε-caprolactone) (PCL), a hydrophobic nonionic polymer, is adopted to enhance the compressive strength of PEG alone.We aimed to investigate the in-vitro response of osteoblast-like cells cultured with porous scaffolds of triblock PEG-PCL-PEG copolymer fabricated by an air pressure-aided deposition system. A desktop air pressure-aided deposition system that involves melting and plotting PEG-PCL-PEG was used to fabricate three-dimensional scaffolds having rectangular pores. The experimental results showed that PEG-PCL-PEG with a molecular weight of 25,000 can be melted and stably deposited through a heating nozzle at an air pressure of 0.3 MPa and no crack occurs after it solidifies. The scaffolds with pre-determined pore size of 400× 420 μm and a porosity of 79 % were fabricated, and their average compressive strength was found to be 18.2 MPa. Osteoblast-like cells, MC3T3-E1, were seeded on fabricated scaffolds to investigate the in-vitro response of cells including toxicity and cellular locomotion. In a culture period of 28 days, the neutral-red stained osteoblasts were found to well distributed in the interior of the scaffold. Furthermore, the cellular attachment and movement in the first 10 h of cell culture were observed with time-lapse microscopy indicating that the porous PEG-PCL-PEG scaffolds fabricated by air pressure-aided deposition system is non-toxicity for osteoblast-like cells.

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors.

PubMed

Stachelscheid, H; Wulf-Goldenberg, A; Eckert, K; Jensen, J; Edsbagge, J; Björquist, P; Rivero, M; Strehl, R; Jozefczuk, J; Prigione, A; Adjaye, J; Urbaniak, T; Bussmann, P; Zeilinger, K; Gerlach, J C

2013-09-01

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models. Copyright © 2012 John Wiley & Sons, Ltd.

Autologous mesenchymal stem cell treatment increased T regulatory cells with no effect on disease activity in two systemic lupus erythematosus patients.

PubMed

Carrion, F; Nova, E; Ruiz, C; Diaz, F; Inostroza, C; Rojo, D; Mönckeberg, G; Figueroa, F E

2010-03-01

Mesenchymal stem cells (MSCs) exert suppressive effects in several disease models including lupus prone mice. However, autologous MSC therapy has not been tested in human systemic lupus erythematosus (SLE). We evaluate the safety and efficacy of bone marrow (BM)-derived MSCs in two SLE patients; the suppressor effect of these cells in-vitro and the change in CD4+CD25+FoxP3+ T regulatory (Treg) cells in response to treatment. Two females (JQ and SA) of 19 and 25 years of age, fulfilling the 1997 American College of Rheumatology (ACR) criteria for SLE were infused with autologous BM-derived MSCs. Disease activity indexes and immunological parameters were assessed at baseline, 1, 2, 7 and 14 weeks. Peripheral blood lymphocyte (PBL) subsets and Treg cells were quantitated by flow cytometry, and MSCs tested for in-vitro suppression of activation and proliferation of normal PBLs. No adverse effects or change in disease activity indexes were noted during 14 weeks of follow-up, although circulating Treg cells increased markedly. Patient MSCs effectively suppressed in-vitro PBL function. However, JQ developed overt renal disease 4 months after infusion. MSC infusion was without adverse effects, but did not modify initial disease activity in spite of increasing CD4+CD25+FoxP3+ cell counts. One patient subsequently had a renal flare. We speculate that the suppressive effects of MSC-induced Treg cells might be dependent on a more inflammatory milieu, becoming clinically evident in patients with higher degrees of disease activity.

Self-assembled pentablock copolymers for selective and sustained gene delivery

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

Zhang, Bingqi

2011-05-15

The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomesmore » of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.« less

Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.

PubMed

Roy, A; Krzykwa, E; Lemieux, R; Néron, S

2001-12-01

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

Organ/body-on-a-chip based on microfluidic technology for drug discovery.

PubMed

Kimura, Hiroshi; Sakai, Yasuyuki; Fujii, Teruo

2018-02-01

Although animal experiments are indispensable for preclinical screening in the drug discovery process, various issues such as ethical considerations and species differences remain. To solve these issues, cell-based assays using human-derived cells have been actively pursued. However, it remains difficult to accurately predict drug efficacy, toxicity, and organs interactions, because cultivated cells often do not retain their original organ functions and morphologies in conventional in vitro cell culture systems. In the μTAS research field, which is a part of biochemical engineering, the technologies of organ-on-a-chip, based on microfluidic devices built using microfabrication, have been widely studied recently as a novel in vitro organ model. Since it is possible to physically and chemically mimic the in vitro environment by using microfluidic device technology, maintenance of cellular function and morphology, and replication of organ interactions can be realized using organ-on-a-chip devices. So far, functions of various organs and tissues, such as the lung, liver, kidney, and gut have been reproduced as in vitro models. Furthermore, a body-on-a-chip, integrating multi organ functions on a microfluidic device, has also been proposed for prediction of organ interactions. We herein provide a background of microfluidic systems, organ-on-a-chip, Body-on-a-chip technologies, and their challenges in the future. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

The prediction of human skin responses by using the combined in vitro fluorescein leakage/Alamar Blue (resazurin) assay.

PubMed

Clothier, Richard; Starzec, Gemma; Pradel, Lionel; Baxter, Victoria; Jones, Melanie; Cox, Helen; Noble, Linda

2002-01-01

A range of cosmetics formulations with human patch-test data were supplied in a coded form, for the examination of the use of a combined in vitro permeability barrier assay and cell viability assay to generate, and then test, a prediction model for assessing potential human skin patch-test results. The target cells employed were of the Madin Darby canine kidney cell line, which establish tight junctions and adherens junctions able to restrict the permeability of sodium fluorescein across the barrier of the confluent cell layer. The prediction model for interpretation of the in vitro assay results included initial effects and the recovery profile over 72 hours. A set of the hand-wash, surfactant-based formulations were tested to generate the prediction model, and then six others were evaluated. The model system was then also evaluated with powder laundry detergents and hand moisturisers: their effects were predicted by the in vitro test system. The model was under-predictive for two of the ten hand-wash products. It was over-predictive for the moisturisers, (two out of six) and eight out of ten laundry powders. However, the in vivo human patch test data were variable, and 19 of the 26 predictions were correct or within 0.5 on the 0-4.0 scale used for the in vivo scores, i.e. within the same variable range reported for the repeat-test hand-wash in vivo data.

A co-culture device with a tunable stiffness to understand combinatorial cell-cell and cell-matrix interactions.

PubMed

Rao, Nikhil; Grover, Gregory N; Vincent, Ludovic G; Evans, Samantha C; Choi, Yu Suk; Spencer, Katrina H; Hui, Elliot E; Engler, Adam J; Christman, Karen L

2013-11-01

Cell behavior on 2-D in vitro cultures is continually being improved to better mimic in vivo physiological conditions by combining niche cues including multiple cell types and substrate stiffness, which are well known to impact cell phenotype. However, no system exists in which a user can systematically examine cell behavior on a substrate with a specific stiffness (elastic modulus) in culture with a different cell type, while maintaining distinct cell populations. We demonstrate the modification of a silicon reconfigurable co-culture system with a covalently linked hydrogel of user-defined stiffness. This device allows the user to control whether two separate cell populations are in contact with each other or only experience paracrine interactions on substrates of controllable stiffness. To illustrate the utility of this device, we examined the role of substrate stiffness combined with myoblast co-culture on adipose derived stem cell (ASC) differentiation and found that the presence of myoblasts and a 10 kPa substrate stiffness increased ASC myogenesis versus co-culture on stiff substrates. As this example highlights, this technology better controls the in vitro microenvironment, allowing the user to develop a more thorough understanding of the combined effects of cell-cell and cell-matrix interactions.

Effect of hyaluronic acid on morphological changes to dentin surfaces and subsequent effect on periodontal ligament cell survival, attachment, and spreading.

PubMed

Mueller, Andrea; Fujioka-Kobayashi, Masako; Mueller, Heinz-Dieter; Lussi, Adrian; Sculean, Anton; Schmidlin, Patrick R; Miron, Richard J

2017-05-01

Hyaluronic acid (HA) is a natural constituent of connective tissues and plays an important role in their development, maintenance, and regeneration. Recently, HA has been shown to improve wound healing. However, no basic in vitro study to date has investigated its mode of action. Therefore, the purpose of this study was to examine morphological changes of dentin surfaces following HA coating and thereafter investigate the influence of periodontal ligament (PDL) cell survival, attachment, and spreading to dentin discs. HA was coated onto dentin discs utilizing either non-cross-linked (HA) or cross-linked (HA cl) delivery systems. Morphological changes to dentin discs were then assessed using scanning electron microscopy (SEM). Thereafter, human PDL cells were seeded under three in vitro conditions including (1) dilution of HA (1:100), (2) dilution of HA (1:10), and (3) HA coated directly to dentin discs. Samples were then investigated for PDL cell survival, attachment, and spreading using a live/dead assay, cell adhesion assay, and SEM imaging, respectively. While control dentin discs demonstrated smooth surfaces both at low and high magnification, the coating of HA altered surface texture of dentin discs by increasing surface roughness. HA cl further revealed greater surface texture/roughness likely due to the cross-linking carrier system. Thereafter, PDL cells were seeded on control and HA coated dentin discs and demonstrated a near 100 % survival rate for all samples demonstrating high biocompatibility of HA at dilutions of both 1:100 and 1:10. Interestingly, non-cross-linked HA significantly increased cell numbers at 8 h, whereas cross-linked HA improved cell spreading as qualitatively assessed by SEM. The results from the present study demonstrate that both carrier systems for HA were extremely biocompatible and demonstrated either improved cell numbers or cell spreading onto dentin discs. Future in vitro and animal research is necessary to further characterize the optimal delivery system of HA for improved clinical use. HA is a highly biocompatible material that may improve PDL cell attachment or spreading on dentin.

Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach.

PubMed

Prestwich, Glenn D

2007-08-15

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.

In vitro transcription of a cloned mouse ribosomal RNA gene.

PubMed Central

Mishima, Y; Yamamoto, O; Kominami, R; Muramatsu, M

1981-01-01

An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system. Images PMID:6278446

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

PubMed Central

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development. PMID:23935837

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

PubMed

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

In Vitro Exposure Systems and Dosimetry Assessment Tools ...

EPA Pesticide Factsheets

In 2009, the passing of The Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP) and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed “modified risk”. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference titled “In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products” to bring together stakeholders representing regulatory agencies, academia, and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapor exposure systems, as well as the various approaches and challenges to quantifying the complex exposures, in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were, 1) Tobacco Smoke And E-Cigarette Aerosols, 2) Air-Liquid Interface-In Vitro Exposure Systems, 3) Dosimetry Approaches For Particles And Vapors; In Vitro Dosimetry Determinations and 4) Exposure Microenvironment/Physiology Of Cells. The two and a half day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will re

Differential biological effects of dehydroepiandrosterone (DHEA) between mouse (B16F10) and human melanoma (BLM) cell lines.

PubMed

Joshi, Kumud; Hassan, Sherif S; Ramaraj, Pandurangan

2017-01-01

Dehydroepiandrosterone (DHEA) is a weak androgen and had been shown to have anti-cancer, anti-adipogenic and anti-inflammatory effects on mouse and other rodent models, but not on humans, suggesting a systemic level difference between mouse and human. Our previous study on DHEA biological functions involving a variety of cell lines, suggested that the functional differences between mouse and human existed even at the cellular level. Hence, using mouse and human melanoma cell models, in-vitro effects of DHEA on cell growth, mechanism of cell death and mechanism of DHEA action were studied. Results indicated a differential biological effects of DHEA between mouse and human melanoma cell lines. These in-vitro studies also suggested that the differential biological effects observed between these two cell lines could be due to the difference in the way DHEA was processed or metabolized inside the cell.

RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

PubMed

Crispo, M; Dos Santos-Neto, P C; Vilariño, M; Mulet, A P; de León, A; Barbeito, L; Menchaca, A

2016-10-01

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro.

PubMed

Tommasi, Stella; Bates, Steven E; Behar, Rachel Z; Talbot, Prue; Besaratinia, Ahmad

2017-10-01

Electronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown. We have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene. We observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Shuttle of lentiviral vectors via transplanted cells in vivo.

PubMed

Blömer, U; Gruh, I; Witschel, H; Haverich, A; Martin, U

2005-01-01

Lentiviral vectors have turned out to be an efficient method for stable gene transfer in vitro and in vivo. Not only do fields of application include cell marking and tracing following transplantation in vivo, but also the stable delivery of biological active proteins for gene therapy. A variety of cells, however, need immediate transplantation after preparation, for example, to prevent cell death, differentiation or de-differentiation. Although these cells are usually washed several times following lentiviral transduction, there may be the risk of viral vector shuttle via transplanted cells resulting in undesired in vivo transduction of recipient cells. We investigated whether infectious lentiviral particles are transmitted via ex vivo lentivirally transduced cells. To this end, we explored potential viral shuttle via ex vivo lentivirally transduced cardiomyocytes in vitro and following transplantation into the brain and peripheral muscle. We demonstrate that, even after extensive washing, infectious viral vector particles can be detected in cell suspensions. Those lentiviral vector particles were able to transduce target cells in transwell experiments. Moreover, transmitted vector particles stably transduced resident cells of the recipient central nervous system and muscle in vivo. Our results of lentiviral vector shuttle via transduced cardiomyocytes are significant for both ex vivo gene therapy and for lentiviral cell tracing, in particular for investigation of stem cell differentiation in transplantation models and co-cultivation systems.

Tailoring microfluidic systems for organ-like cell culture applications using multiphysics simulations

NASA Astrophysics Data System (ADS)

Hagmeyer, Britta; Schütte, Julia; Böttger, Jan; Gebhardt, Rolf; Stelzle, Martin

2013-03-01

Replacing animal testing with in vitro cocultures of human cells is a long-term goal in pre-clinical drug tests used to gain reliable insight into drug-induced cell toxicity. However, current state-of-the-art 2D or 3D cell cultures aiming at mimicking human organs in vitro still lack organ-like morphology and perfusion and thus organ-like functions. To this end, microfluidic systems enable construction of cell culture devices which can be designed to more closely resemble the smallest functional unit of organs. Multiphysics simulations represent a powerful tool to study the various relevant physical phenomena and their impact on functionality inside microfluidic structures. This is particularly useful as it allows for assessment of system functions already during the design stage prior to actual chip fabrication. In the HepaChip®, dielectrophoretic forces are used to assemble human hepatocytes and human endothelial cells in liver sinusoid-like structures. Numerical simulations of flow distribution, shear stress, electrical fields and heat dissipation inside the cell assembly chambers as well as surface wetting and surface tension effects during filling of the microchannel network supported the design of this human-liver-on-chip microfluidic system for cell culture applications. Based on the device design resulting thereof, a prototype chip was injection-moulded in COP (cyclic olefin polymer). Functional hepatocyte and endothelial cell cocultures were established inside the HepaChip® showing excellent metabolic and secretory performance.

Biotechnological applications in in vitro plant regeneration studies of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

PubMed

Kumar, Pankaj; Srivastava, Dinesh Kumar

2016-04-01

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.

Inhibition of aberrant circulating Tfh cell proportions by corticosteroids in patients with systemic lupus erythematosus.

PubMed

Feng, Xuebing; Wang, Dandan; Chen, Jingjing; Lu, Lin; Hua, Bingzhu; Li, Xia; Tsao, Betty P; Sun, Lingyun

2012-01-01

To observe the proportion of peripheral T follicular helper (Tfh) cells in patients with systemic lupus erythematosus (SLE) and to assess the role of steroids on Tfh cells from SLE patients. Peripheral blood mononuclear cells (PBMCs) from 42 SLE patients and 22 matched healthy subjects were collected to assess proportions of circulating CXCR5(+)PD1(+)/CD4(+) T cells (Tfh), CD4(+)CCR6(+) T cells (Th17-like) and CD19(+)CD138(+) plasma cells by flow cytometry. 8 of the patients had their blood redrawn within one week after receiving methylprednisolone pulse treatment. Disease activity was evaluated by SLE disease activity index. To test the effect of IL-21 and corticosteroids on Tfh cells in vitro, PBMCs harvested from another 15 SLE patients were cultured with medium, IL-21, or IL-21+ dexamethasone for 24 hours and 72 hours. PBMCs from an independent 23 SLE patients were cultured with different concentrations of dexamethasone for 24 hours. Compared to normal controls, percentages of circulating Tfh cells, but not Th17 cells, were elevated in SLE patients and correlated with disease activity. Proportions of Tfh cells in SLE patients were positively correlated with those of plasma cells and serum levels of antinuclear antibodies. After methylprednisolone pulse treatment, both percentages and absolute numbers of circulating Tfh cells were significantly decreased. In vitro cultures showed an increase of Tfh cell proportion after IL-21 stimulation that was totally abolished by the addition of dexamethasone. Both 0.5 and 1 µM dexamethasone decreased Tfh cells dose dependently (overall p = 0.013). We demonstrated that elevated circulating Tfh cell proportions in SLE patients correlated with their disease activities, and circulating levels of plasma cells and ANA. Corticosteroids treatment down-regulated aberrant circulating Tfh cell proportions both in vivo and in vitro, making Tfh cells a new treatment target for SLE patients.

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

PubMed

Hiroishi, S; Sugie, K; Yoshida, T; Morimoto, J; Taniguchi, Y; Imai, S; Kurebayashi, J

2001-06-26

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

PubMed Central

SUGIMURA, Satoshi; AKAI, Tomonori; HASHIYADA, Yutaka; AIKAWA, Yoshio; OHTAKE, Masaki; MATSUDA, Hideo; KOBAYASHI, Shuji; KOBAYASHI, Eiji; KONISHI, Kazuyuki; IMAI, Kei

2012-01-01

Abstract To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density. PMID:23154384

Different in vitro cellular responses to tamoxifen treatment in polydimethylsiloxane-based devices compared to normal cell culture.

PubMed

Wang, Lingyu; Yu, Linfen; Grist, Samantha; Cheung, Karen C; Chen, David D Y

2017-11-15

Cell culture systems based on polydimethylsiloxane (PDMS) microfluidic devices offer great flexibility because of their simple fabrication and adaptability. PDMS devices also make it straightforward to set up parallel experiments and can facilitate process automation, potentially speeding up the drug discovery process. However, cells grown in PDMS-based systems can develop in different ways to those grown with conventional culturing systems because of the differences in the containers' surfaces. Despite the growing number of studies on microfluidic cell culture devices, the differences in cellular behavior in PDMS-based devices and normal cell culture systems are poorly characterized. In this work, we investigated the proliferation and autophagy of MCF7 cells cultured in uncoated and Parylene-C coated PDMS wells. Using a quantitative method combining solid phase extraction and liquid chromatography mass spectrometry we developed, we showed that Tamoxifen uptake into the surfaces of uncoated PDMS wells can change the drug's effective concentration in the culture medium, affecting the results of Tamoxifen-induced autophagy and cytotoxicity assays. Such changes must be carefully analyzed before transferring in vitro experiments from a traditional culture environment to a PDMS-based microfluidic system. We also found that cells cultured in Parylene-C coated PDMS wells showed similar proliferation and drug response characteristics to cells cultured in standard polystyrene (PS) plates, indicating that Parylene-C deposition offers an easy way of limiting the uptake of small molecules into porous PDMS materials and significantly improves the performance of PDMS-based device for cell related research. Copyright © 2017 Elsevier B.V. All rights reserved.

Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro.

PubMed

Cipriano, Madalena; Freyer, Nora; Knöspel, Fanny; Oliveira, Nuno G; Barcia, Rita; Cruz, Pedro E; Cruz, Helder; Castro, Matilde; Santos, Jorge M; Zeilinger, Katrin; Miranda, Joana P

2017-04-01

3D cultures of human stem cell-derived hepatocyte-like cells (HLCs) have emerged as promising models for short- and long-term maintenance of hepatocyte phenotype in vitro cultures by better resembling the in vivo environment of the liver and consequently increase the translational value of the resulting data. In this study, the first stage of hepatic differentiation of human neonatal mesenchymal stem cells (hnMSCs) was performed in 2D monolayer cultures for 17 days. The second stage was performed by either maintaining cells in 2D cultures for an extra 10 days, as control, or alternatively cultured in 3D as self-assembled spheroids or in multicompartment membrane bioreactor system. All systems enabled hnMSC differentiation into HLCs as shown by positive immune staining of hepatic markers CK-18, HNF-4α, albumin, the hepatic transporters OATP-C and MRP-2 as well as drug-metabolizing enzymes like CYP1A2 and CYP3A4. Similarly, all models also displayed relevant glucose, phase I and phase II metabolism, the ability to produce albumin and to convert ammonia into urea. However, EROD activity and urea production were increased in both 3D systems. Moreover, the spheroids revealed higher bupropion conversion, whereas bioreactor showed increased albumin production and capacity to biotransform diclofenac. Additionally, diclofenac resulted in an IC 50 value of 1.51 ± 0.05 and 0.98 ± 0.03 in 2D and spheroid cultures, respectively. These data suggest that the 3D models tested improved HLC maturation showing a relevant biotransformation capacity and thus provide more appropriate reliable models for mechanistic studies and more predictive systems for in vitro toxicology applications.

Purification and cultivation of human pituitary growth hormone secreting cells

NASA Technical Reports Server (NTRS)

Hymer, W. C.

1978-01-01

The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system.

Purpose-driven biomaterials research in liver-tissue engineering.

PubMed

Ananthanarayanan, Abhishek; Narmada, Balakrishnan Chakrapani; Mo, Xuejun; McMillian, Michael; Yu, Hanry

2011-03-01

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications. Copyright © 2010 Elsevier Ltd. All rights reserved.

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.

In vitro DNA SCRaMbLE.

PubMed

Wu, Yi; Zhu, Rui-Ying; Mitchell, Leslie A; Ma, Lu; Liu, Rui; Zhao, Meng; Jia, Bin; Xu, Hui; Li, Yun-Xiang; Yang, Zu-Ming; Ma, Yuan; Li, Xia; Liu, Hong; Liu, Duo; Xiao, Wen-Hai; Zhou, Xiao; Li, Bing-Zhi; Yuan, Ying-Jin; Boeke, Jef D

2018-05-22

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a β-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

Cell-Extracellular Matrix Mechanobiology: Forceful Tools and Emerging Needs for Basic and Translational Research.

PubMed

Holle, Andrew W; Young, Jennifer L; Van Vliet, Krystyn J; Kamm, Roger D; Discher, Dennis; Janmey, Paul; Spatz, Joachim P; Saif, Taher

2018-01-10

Extracellular biophysical cues have a profound influence on a wide range of cell behaviors, including growth, motility, differentiation, apoptosis, gene expression, adhesion, and signal transduction. Cells not only respond to definitively mechanical cues from the extracellular matrix (ECM) but can also sometimes alter the mechanical properties of the matrix and hence influence subsequent matrix-based cues in both physiological and pathological processes. Interactions between cells and materials in vitro can modify cell phenotype and ECM structure, whether intentionally or inadvertently. Interactions between cell and matrix mechanics in vivo are of particular importance in a wide variety of disorders, including cancer, central nervous system injury, fibrotic diseases, and myocardial infarction. Both the in vitro and in vivo effects of this coupling between mechanics and biology hold important implications for clinical applications.

Characterization and isolation of highly purified porcine satellite cells

PubMed Central

Ding, Shijie; Wang, Fei; Liu, Yan; Li, Sheng; Zhou, Guanghong; Hu, Ping

2017-01-01

Pig is an important food source and an excellent system to model human diseases. Careful characterization of the swine skeletal muscle stem cells (satellite cells) will shed lights on generation of swine skeletal muscle disease model and efficient production of porcine meat for the food industry. Paired box protein 7 (Pax7) is a highly conserved transcription factor shared by satellite cells from various species. However, the sequence of Pax7 has not been characterized in pig. The lack of method to isolate highly purified satellite cells hinders the thorough characterization of the swine satellite cells. Here we found molecular markers for swine satellite cells and revealed that the porcine satellite cells were heterogeneous in various pieces of skeletal muscle. We further developed a method to isolate highly purified satellite cells directly from porcine muscles using fluorescence-activated cell sorting. We next characterized the proliferation and differentiation abilities of isolated satellite cells in vitro; and found that long-term culturing of satellite cells in vitro led to stemness loss. PMID:28417015

Two cytotoxic sesquiterpene lactones from the leaves of Xanthium strumarium and their in vitro inhibitory activity on farnesyltransferase.

PubMed

Kim, Young Sup; Kim, Jeoung Seob; Park, Sung-Hee; Choi, Sang-Un; Lee, Chong Ock; Kim, Seong-Kie; Kim, Young-Kyoon; Kim, Sung Hoon; Ryu, Shi Yong

2003-04-01

Two xanthanolide sesquiterpene lactones, 8- epi-xanthatin (1) and 8- epi-xanthatin epoxide (2), isolated from the leaves of Xanthium strumarium (Compositae), demonstrated a significant inhibition on the proliferation of cultured human tumor cells, i. e., A549 (non-small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nervous system) and HCT-15 (colon) in vitro. They were also found to inhibit the farnesylation process of human lamin-B by farnesyltransferase (FTase), in a dose-dependent manner in vitro (IC 50 value was calculated as 64 and 58 microM, respectively). Due to the relatively high concentrations of 1 and 2 required to obtain an FTase inhibition as compared with those necessary for a cytotoxic effect on tumor cells, it remains unclear whether a relationship between these two activities exists.

In vitro cytotoxicity and differential cellular sensitivity of derivatives of diamino acids. II. N1-methyl, N1-allyl, N1-(2-chloroethyl) and N1-propargyl nitrosoureas.

PubMed

Dulude, H; Salvador, R; Gallant, G

1995-01-01

The in vitro cytotoxicity and differential cellular sensitivity of a series of new N1-methyl, N1-allyl, N1-2-chloroethyl and N1-propargyl nitrosourea derivatives of diamino acids were determined in the National Cancer Institute's primary antitumor drug screen. The compounds tested showed an in vitro anticancer activity similar to commercialized nitrosoureas such as CCNU, BCNU, MeCCNU, chlorozotocin, streptozotocin and PCNU. The alkylating moiety of the nitrosoureas seems to play a role in the general selectivity of our compounds. The N1-methyl and N1-2-chloroethyl nitrosourea derivatives are more selective for central nervous system cell lines, the N1-allyl nitrosourea derivatives are more selective for lung cancer cell lines and the N1-propargyl nitrosoureas are more selective for leukemia cell lines.

Development of a rectal nicotine delivery system for the treatment of ulcerative colitis.

PubMed

Dash, A K; Gong, Z; Miller, D W; Huai-Yan, H; Laforet, J

1999-11-10

The aims of this investigation were: i. to develop a rectal nicotine delivery system with bioadhesives for the treatment of ulcerative colitis and ii. to evaluate nicotine transport and cytotoxicity of the delivery system using Caco-2 cell culture systems. Rectal nicotine suppository formulations were prepared in semi-synthetic glyceride bases (Suppocire AM and AI, Gattefosse Inc.) by fusion method. The in vitro release of nicotine was carried out in modified USP dissolution apparatus 1. Differential scanning calorimetry (DSC) and powder X-ray diffraction were used to study the polymorphic changes if any in the formulations. An LC method was used for the assay of nicotine. The effect of bioadhesives (glyceryl monooleate (GMO), and Carbopol) on the nicotine flux was evaluated using Caco-2 cell permeability studies and Caco-2 cell viability was determined using the MTT toxicity assay. In vitro release studies indicated that the low melting AI base was superior to that of the AM base. Presence of GMO in the formulation enhanced the release of nicotine whereas Carbopol showed an opposite effect. The enhanced release of nicotine in the presence of GMO was found to be partly due to the melting point lowering effect of this compound. Caco-2 cell absorption studies showed that there was a decrease in the flux of nicotine in the presence of both the bioadhesives. The flux of the fluorescein marker which is used to study the integrity of the cell monolayers was found to be slightly higher only in the presence of 10% (w/w) Carbopol. Nicotine, Carbopol, and GMO do not have any cytotoxic effect on these cell monolayers within the concentration range used in the formulations. Rectal nicotine formulations containing bioadhesives were developed and characterized. Both in vitro release and cell culture studies have indicated that one can manipulate the nicotine release from these rectal delivery systems by incorporation of various bioadhesives or the use of different bases in the formulation. Nicotine concentration below 2% (w/v) and bioadhesive concentration below 10% (w/w) do not have any cytotoxic effect on Caco-2 cells.

In vitro-ex vivo correlations between a cell-laden hydrogel and mucosal tissue for screening composite delivery systems

PubMed Central

Blakney, Anna K.; Little, Adam B.; Jiang, Yonghou; Woodrow, Kim A.

2017-01-01

Composite delivery systems where drugs are electrospun in different layers and vary the drug stacking-order are posited to affect bioavailability. We evaluated how the formulation characteristics of both burst- and sustained-release electrospun fibers containing three physicochemically diverse drugs: dapivirine (DPV), maraviroc (MVC) and tenofovir (TFV) affect in vitro and ex vivo release. We developed a poly(hydroxyethyl methacrylate) (pHEMA) hydrogel release platform for the rapid, inexpensive in vitro evaluation of burst- and sustained-release topical or dermal drug delivery systems with varying microarchitecture. We investigated properties of the hydrogel that could recapitulate ex vivo release into nonhuman primate vaginal tissue. Using a DMSO extraction protocol and HPLC analysis, we achieved >93% recovery from the hydrogels and >88% recovery from tissue explants for all three drugs. We found that DPV loading, but not stacking order (layers of fiber containing a single drug) or microarchitecture (layers with isolated drug compared to all drugs in the same layer) impacted the burst release in vitro and ex vivo. Our burst-release formulations showed a correlation for DPV accumulation between the hydrogel and tissue (R2=0.80), but the correlation was not significant for MVC or TFV. For the sustained release formulations, the PLGA/PCL content did not affect TFV release in vitro or ex vivo. Incorporation of cells into the hydrogel matrix improved the correlation between hydrogel and tissue explant release for TFV. We expect that this hydrogel tissue mimic maybe a promising preclinical model to evaluate topical or transdermal drug delivery systems with complex microarchitectures. PMID:28222612

The stentable in vitro artery: an instrumented platform for endovascular device development and optimization.

PubMed

Antoine, Elizabeth E; Cornat, François P; Barakat, Abdul I

2016-12-01

Although vascular disease is a leading cause of mortality, in vitro tools for controlled, quantitative studies of vascular biological processes in an environment that reflects physiological complexity remain limited. We developed a novel in vitro artery that exhibits a number of unique features distinguishing it from tissue-engineered or organ-on-a-chip constructs, most notably that it allows deployment of endovascular devices including stents, quantitative real-time tracking of cellular responses and detailed measurement of flow velocity and lumenal shear stress using particle image velocimetry. The wall of the stentable in vitro artery consists of an annular collagen hydrogel containing smooth muscle cells (SMCs) and whose lumenal surface is lined with a monolayer of endothelial cells (ECs). The system has in vivo dimensions and physiological flow conditions and allows automated high-resolution live imaging of both SMCs and ECs. To demonstrate proof-of-concept, we imaged and quantified EC wound healing, SMC motility and altered shear stresses on the endothelium after deployment of a coronary stent. The stentable in vitro artery provides a unique platform suited for a broad array of research applications. Wide-scale adoption of this system promises to enhance our understanding of important biological events affecting endovascular device performance and to reduce dependence on animal studies. © 2016 The Author(s).

New bioactive motifs and their use in functionalized self-assembling peptides for NSC differentiation and neural tissue engineering

NASA Astrophysics Data System (ADS)

Gelain, F.; Cigognini, D.; Caprini, A.; Silva, D.; Colleoni, B.; Donegá, M.; Antonini, S.; Cohen, B. E.; Vescovi, A.

2012-04-01

Developing functionalized biomaterials for enhancing transplanted cell engraftment in vivo and stimulating the regeneration of injured tissues requires a multi-disciplinary approach customized for the tissue to be regenerated. In particular, nervous tissue engineering may take a great advantage from the discovery of novel functional motifs fostering transplanted stem cell engraftment and nervous fiber regeneration. Using phage display technology we have discovered new peptide sequences that bind to murine neural stem cell (NSC)-derived neural precursor cells (NPCs), and promote their viability and differentiation in vitro when linked to LDLK12 self-assembling peptide (SAPeptide). We characterized the newly functionalized LDLK12 SAPeptides via atomic force microscopy, circular dichroism and rheology, obtaining nanostructured hydrogels that support human and murine NSC proliferation and differentiation in vitro. One functionalized SAPeptide (Ac-FAQ), showing the highest stem cell viability and neural differentiation in vitro, was finally tested in acute contusive spinal cord injury in rats, where it fostered nervous tissue regrowth and improved locomotor recovery. Interestingly, animals treated with the non-functionalized LDLK12 had an axon sprouting/regeneration intermediate between Ac-FAQ-treated animals and controls. These results suggest that hydrogels functionalized with phage-derived peptides may constitute promising biomimetic scaffolds for in vitro NSC differentiation, as well as regenerative therapy of the injured nervous system. Moreover, this multi-disciplinary approach can be used to customize SAPeptides for other specific tissue engineering applications.Developing functionalized biomaterials for enhancing transplanted cell engraftment in vivo and stimulating the regeneration of injured tissues requires a multi-disciplinary approach customized for the tissue to be regenerated. In particular, nervous tissue engineering may take a great advantage from the discovery of novel functional motifs fostering transplanted stem cell engraftment and nervous fiber regeneration. Using phage display technology we have discovered new peptide sequences that bind to murine neural stem cell (NSC)-derived neural precursor cells (NPCs), and promote their viability and differentiation in vitro when linked to LDLK12 self-assembling peptide (SAPeptide). We characterized the newly functionalized LDLK12 SAPeptides via atomic force microscopy, circular dichroism and rheology, obtaining nanostructured hydrogels that support human and murine NSC proliferation and differentiation in vitro. One functionalized SAPeptide (Ac-FAQ), showing the highest stem cell viability and neural differentiation in vitro, was finally tested in acute contusive spinal cord injury in rats, where it fostered nervous tissue regrowth and improved locomotor recovery. Interestingly, animals treated with the non-functionalized LDLK12 had an axon sprouting/regeneration intermediate between Ac-FAQ-treated animals and controls. These results suggest that hydrogels functionalized with phage-derived peptides may constitute promising biomimetic scaffolds for in vitro NSC differentiation, as well as regenerative therapy of the injured nervous system. Moreover, this multi-disciplinary approach can be used to customize SAPeptides for other specific tissue engineering applications. Electronic supplementary information (ESI) available: Supporting methods and data about CD spectral analysis of SAPeptide solutions (Fig. S1), neural differentiation of murine and human NSCs (Fig. S2) on SAPeptide scaffolds, and their statistical analysis (Table S1). See DOI: 10.1039/c2nr30220a

In vitro and ex vivo strategies for intracellular delivery

NASA Astrophysics Data System (ADS)

Stewart, Martin P.; Sharei, Armon; Ding, Xiaoyun; Sahay, Gaurav; Langer, Robert; Jensen, Klavs F.

2016-10-01

Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.

Vincristine-sulphate-loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system.

PubMed

Thakkar, Hetal Paresh; Baser, Amit Kumar; Parmar, Mayur Prakashbhai; Patel, Ketul Harshadbhai; Ramachandra Murthy, Rayasa

2012-06-01

Vincristine-sulfate-loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate-coated liposomes show higher cell uptake than uncoated liposomes.

Involvement of DMT1 +IRE in the transport of lead in an in vitro BBB model.

PubMed

Wang, Qiang; Luo, Wenjing; Zhang, Wenbin; Liu, Mingchao; Song, Haifeng; Chen, Jingyuan

2011-06-01

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB). The BBB is particularly vulnerable to lead (Pb) insults. This study was designed to test the hypothesis that divalent metal transporter 1 (DMT1), which is a divalent cation membrane transporter, was involved in transcellular transport across the BBB. An in vitro BBB model, which was a co-culture system of human umbilical vascular endothelial cells (ECV304) and rat glioma cells (C6), was established. Transendothelial electrical resistance (TEER) and fluoresceinisothiocyanate (FITC)-dextran permeability results showed that Pb exposure at the tested concentrations had no significant effects on intercellular tightness. Pb transport displayed properties that were associated with iron response element (IRE) positive isoform of DMT1. Accordingly, Pb transport was significantly blocked by iron (Fe). Moreover, ECV304 cells that were depleted of Fe with the chelator deferoxamine (DFO) demonstrated increased Pb transport. By transfecting ECV-304 cells with a DMT1 expression vector, overexpression of DMT1 promoted an increase in Pb transport. Treatment of ECV304 cells with DMT1 antisense oligonucleotides (ASONs) MA1 significantly inhibited the transport of Pb. Our results suggest that Pb is transported in the in vitro BBB model by a transporter with biochemical properties similar to those of the DMT1 IRE-positive isoform. Copyright © 2009 Elsevier Ltd. All rights reserved.

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue.

PubMed

Kaur, Pavinder; Ward, Brenda; Saha, Baisakhi; Young, Lillian; Groshen, Susan; Techy, Geza; Lu, Yani; Atkinson, Roscoe; Taylor, Clive R; Ingram, Marylou; Imam, S Ashraf

2011-12-01

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.

Cell-Cell Interactions during pollen tube guidance

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

Daphne Preuss

The long-term goal of this research is to identify the signaling molecules that mediate plant cell-cell interactions during pollination. The immediate goals of this project are to perform genetic and molecular analysis of pollen tube guidance. Specifically, we proposed to: 1. Characterize the pistil components that direct pollen tube navigation using the Arabidopsis thaliana in vitro pollen tube guidance system 2. Identify pistil signals that direct pollen tube guidance by a) using microarrays to profile gene expression in developing pistils, and b) employing proteomics and metabolomics to isolate pollen tube guidance signals. 3. Explore the genetic basis of natural variationmore » in guidance signals, comparing the in vitro interactions between pollen and pistils from A. thaliana and its close relatives.« less

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay: Book Chapter

EPA Science Inventory

There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adher...

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay-Book Chapter*

EPA Science Inventory

There are thousands of environmental chemicals for which there is limited toxicological information, motivating the development and application of in vitro systems to profile the biological effects of xenobiotic exposure and predict their potential developmental hazard. An adhere...

Engineering Bony Hybrid Organs In Vitro

DTIC Science & Technology

2006-11-01

microfluidic cell culture system using elastomeric channels and Braille displays (Gu et al., 2004; Futai et al., 2004). We have now further developed this...Takayama S, 2004: Computerized microfluidic cell culture using elastomeric channels and braille displays. Proc. Natl. Acad. Sci. USA 101, 15861-15866

Long-term maintenance of luteinizing hormone-responsive testosterone formation by primary rat Leydig cells in vitro.

PubMed

Wang, Yiyan; Huang, Shengsong; Wang, Zhao; Chen, Fenfen; Chen, Panpan; Zhao, Xingxing; Lin, Han; Ge, Renshan; Zirkin, Barry; Chen, Haolin

2018-04-24

The inability of cultured primary Leydig cells to maintain luteinizing hormone (LH)-responsive testosterone formation in vitro for more than 3-5 days has presented a major challenge in testing trophic effects of regulatory factors or environmental toxicants. Our primary objective was to establish culture conditions sufficient to maintain LH-responsive testosterone formation by Leydig cells for at least a month. When isolated rat adult Leydig cells were cultured in DMEM/F12 and M199 culture medium containing insulin (10μg/ml), PDGFAA (10 ng/ml), lipoprotein (0.25 mg/ml), horse serum (1%) and a submaximal concentration of LH (0.2 ng/ml), the cells retained the ability to produce testosterone in vitro for at least 4 weeks. By using the longer-term culture conditions of this system, we were able to detect suppressive effects on testosterone production by low levels of the toxicant MEHP (mono-(2-ethylhexyl) phthalate), an active metabolite of the plasticizer DEHP, that were not detected by short-term culture. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel approach for in vitro studies applying electrical fields to cell cultures by transformer-like coupling.

PubMed

Hess, R; Neubert, H; Seifert, A; Bierbaum, S; Hart, D A; Scharnweber, D

2012-12-01

The purpose of this study was to develop a new apparatus for in vitro studies applying low frequency electrical fields to cells without interfering side effects like biochemical reactions or magnetic fields which occur in currently available systems. We developed a non-invasive method by means of the principle of transformer-like coupling where the magnetic field is concentrated in a toroid and, therefore, does not affect the cell culture. Next to an extensive characterization of the electrical field parameters, initial cell culture studies have focused on examining the response of bone marrow-derived human mesenchymal stem cells (MSCs) to pulsed electrical fields. While no significant differences in the proliferation of human MSCs could be detected, significant increases in ALP activity as well as in gene expression of other osteogenic markers were observed. The results indicate that transformer-like coupled electrical fields can be used to influence osteogenic differentiation of human MSCs in vitro and can pose a useful tool in understanding the influence of electrical fields on the cellular and molecular level.

In vitro non-homologous DNA end joining assays—The 20th anniversary

PubMed Central

Pastwa, Elzbieta; Somiari, Richard I.; Malinowski, Mariusz; Somiari, Stella B.; Winters, Thomas A.

2010-01-01

DNA double-strand breaks (DSBs) are the most serious forms of DNA damage in cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to mutations or cell death, and consequently, to cancer predisposition. In human cells non-homologous DNA end joining (NHEJ) is the main repair mechanism of these breaks. Systems for DNA end joining study have been developing during the last 20 years. New assays have some advantages over earlier in vitro DSBs repair assays because they are less time-consuming, allow the use of clinical material and examination of the joining DNA ends produced physiologically in mammalian cells. Proteins involved in NHEJ repair pathway can serve as biomarkers or molecular targets for anticancer drugs. Results of studies on NHEJ in cancer could help to select potent repair inhibitors that may selectively sensitize tumor cells to ionizing radiation (IR) and chemotherapy. Here, we review the principles and practice of in vitro NHEJ assays and provide some insights into the future prospects of this assay in cancer diagnosis and treatment. PMID:19110069

Antioxidant effects of aminosalicylates and potential new drugs for inflammatory bowel disease: assessment in cell-free systems and inflamed human colorectal biopsies.

PubMed

Simmonds, N J; Millar, A D; Blake, D R; Rampton, D S

1999-03-01

The therapeutic efficacy of 5-aminosalicylic acid in inflammatory bowel disease may be related to its antioxidant properties. To compare in vitro the antioxidant effects of conventional drugs (5-aminosalicylic acid, corticosteroids, metronidazole), with new aminosalicylates (4-aminosalicylic acid, balsalazide) and other potential therapies (ascorbate, N-acetylcysteine, glutathione, verapamil). Compounds were assessed for efficacy in reducing the in vitro production of reactive oxygen species by cell-free systems (using xanthine/xanthine oxidase, with or without myeloperoxidase) and by colorectal biopsies from patients with ulcerative colitis using luminol-amplified chemiluminescence. 5-aminosalicylic acid and balsalazide were more potent antioxidants than 4-aminosalicylic acid or N-acetyl-5-aminosalicylic acid in cell-free systems. 5-aminosalicylic acid (20 mM) and balsalazide (20 mM) inhibited rectal biopsy chemiluminescence by 93% and 100%, respectively, compared with only 59% inhibition by 4-aminosalicylic acid (20 mM). Hydrocortisone, metronidazole and verapamil had no significant effect on chemiluminescence in any system. Ascorbate (20 mM) inhibited chemiluminescence by 100% in cell-free systems and by 60% in rectal biopsies. N-acetyl cysteine (10 mM), and both oxidized and reduced glutathione (10 mM), completely inhibited chemiluminescence in cell-free systems, but not with rectal biopsies. The antioxidant effects of compounds varies between cell-free systems and inflamed colorectal biopsies. The effect of drugs on the chemiluminescence produced by these two assay systems is useful for screening potentially new antioxidant treatments for inflammatory bowel disease. Ascorbate seems worth further study as a novel therapy.

Novel Nano-Therapeutic Approach Actively Targets Human Ovarian Cancer Stem Cells after Xenograft into Nude Mice.

PubMed

Abou-ElNaga, Amoura; Mutawa, Ghada; El-Sherbiny, Ibrahim M; Abd-ElGhaffar, Hassan; Allam, Ahmed A; Ajarem, Jamaan; Mousa, Shaker A

2017-04-12

The power of tumorigenesis, chemo-resistance and metastasis in malignant ovarian tumors resides in a tiny population of cancer cells known as ovarian cancer stem cells (OCSCs). Developing nano-therapeutic targeting of OCSCs is considered a great challenge. The potential use of poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) was investigated as a drug delivery system for paclitaxel (PTX) against OCSCs in vitro and in vivo. PTX-loaded PLGA NPs were prepared by an emulsion solvent evaporation method, supported by incorporation of folic acid (FA) as the ligand. NPs were characterized for size, surface morphology, drug loading, and encapsulation efficiency. In vitro cytotoxicity of PTX-loaded FA/PLGA NPs was tested against OCSCs with MTT assay. In vivo anti-tumoral efficiency and active targeting potential of prepared NPs against tumors in nude mice were investigated. In vitro results revealed that IC 50 of PTX was significantly reduced after loading on PLGA NPs. On the other hand, in vivo results showed that PLGA NPs enhanced the tumor suppression efficiency of PTX. Investigation with real time quantitative PCR analysis revealed the limiting expression of chemo-resistant genes ( ABCG2 and MDR1 ) after applying PLGA NPs as a drug delivery system for PTX. Histopathological examination of tumors showed the effective biological influence of PTX-loaded FA/PLGA NPs through the appearance of reactive lymphoid follicles. Targeting potential of PTX was activated by FA/PLGA NPs through significant preservation of body weight ( p < 0.0001) and minimizing the systemic toxicity in healthy tissues. Immunohistochemical investigation revealed a high expression of apoptotic markers in tumor tissue, supporting the targeting effect of FA/PLGA NPs. A drug delivery system based on FA/PLGA NPs can enhance PTX's in vitro cytotoxicity and in vivo targeting potential against OCSCs.

In vitro bioavailability and cellular bioactivity studies of flavonoids and flavonoid-rich plant extracts: questions, considerations and future perspectives.

PubMed

Gonzales, Gerard Bryan

2017-08-01

In vitro techniques are essential in elucidating biochemical mechanisms and for screening a wide range of possible bioactive candidates. The number of papers published reporting in vitro bioavailability and bioactivity of flavonoids and flavonoid-rich plant extracts is numerous and still increasing. However, even with the present knowledge on the bioavailability and metabolism of flavonoids after oral ingestion, certain inaccuracies still persist in the literature, such as the use of plant extracts to study bioactivity towards vascular cells. There is therefore a need to revisit, even question, these approaches in terms of their biological relevance. In this review, the bioavailability of flavonoid glycosides, the use of cell models for intestinal absorption and the use of flavonoid aglycones and flavonoid-rich plant extracts in in vitro bioactivity studies will be discussed. Here, we focus on the limitations of current in vitro systems and revisit the validity of some in vitro approaches, and not on the detailed mechanism of flavonoid absorption and bioactivity. Based on the results in the review, there is an apparent need for stricter guidelines on publishing data on in vitro data relating to the bioavailability and bioactivity of flavonoids and flavonoid-rich plant extracts.

A three-dimensional cell culture system as an in vitro canine mammary carcinoma model for the expression of connective tissue modulators.

PubMed

Cardoso, T C; Sakamoto, S S; Stockmann, D; Souza, T F B; Ferreira, H L; Gameiro, R; Vieira, F V; Louzada, M J Q; Andrade, A L; Flores, E F

2017-06-01

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two-dimensional (2D) and three-dimensional (3D) conditions. The 3D was performed in six-well AlgiMatrix™ (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50-125 µm for CC and 175-200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases-1, -2, -9 and -13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E-cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)-derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model. © 2016 John Wiley & Sons Ltd.

Impact of the Sensory Neurons on Melanoma Growth In Vivo

PubMed Central

Tapias, Victor; Watkins, Simon C.; Ma, Yang; Shurin, Michael R.; Shurin, Galina V.

2016-01-01

Nerve endings are often identified within solid tumors, but their impact on the tumor growth and progression remains poorly understood. Emerging data suggests that the central nervous system may affect cancer development and spreading via the hypothalamic-pituitary-adrenal axis and autonomous nervous system. However, the role of the afferent sensory neurons in tumor growth is unclear, except some reports on perineural invasion in prostate and pancreatic cancer and cancer-related pain syndrome. Here, we provide the results of primary testing of the concept that the interaction between melanoma cells and sensory neurons may induce the formation of tumor-supporting microenvironment via attraction of immune regulatory cells by the tumor-activated dorsal root ganglion (DRG) neurons. We report that despite DRG cells not directly up-regulating proliferation of melanoma cells in vitro, presence of DRG neurons allows tumors to grow significantly faster in vivo. This effect has been associated with increased production of chemokines by tumor-activated DRG neurons and attraction of myeloid-derived suppressor cells both in vitro and in vivo. These initial proof-of-concept results justify further investigations of the sensory (afferent) nervous system in the context of tumorigenesis and the local protumorigenic immunoenvironment. PMID:27227315

Glia-neuron interactions in neurological diseases: Testing non-cell autonomy in a dish.

PubMed

Meyer, Kathrin; Kaspar, Brian K

2017-02-01

For the past century, research on neurological disorders has largely focused on the most prominently affected cell types - the neurons. However, with increasing knowledge of the diverse physiological functions of glial cells, their impact on these diseases has become more evident. Thus, many conditions appear to have more complex origins than initially thought. Since neurological pathologies are often sporadic with unknown etiology, animal models are difficult to create and might only reflect a small portion of patients in which a mutation in a gene has been identified. Therefore, reliable in vitro systems to studying these disorders are urgently needed. They might be a pre-requisite for improving our understanding of the disease mechanisms as well as for the development of potential new therapies. In this review, we will briefly summarize the function of different glial cell types in the healthy central nervous system (CNS) and outline their implication in the development or progression of neurological conditions. We will then describe different types of culture systems to model non-cell autonomous interactions in vitro and evaluate advantages and disadvantages. This article is part of a Special Issue entitled SI: Exploiting human neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

Concise Review: Adult Mesenchymal Stem Cells, Adult Neural Crest Stem Cells, and Therapy of Neurological Pathologies: A State of Play

PubMed Central

Neirinckx, Virginie; Coste, Cécile; Rogister, Bernard

2013-01-01

Adult stem cells are endowed with in vitro multilineage differentiation abilities and constitute an attractive autologous source of material for cell therapy in neurological disorders. With regard to lately published results, the ability of adult mesenchymal stem cells (MSCs) and neural crest stem cells (NCSCs) to integrate and differentiate into neurons once inside the central nervous system (CNS) is currently questioned. For this review, we collected exhaustive data on MSC/NCSC neural differentiation in vitro. We then analyzed preclinical cell therapy experiments in different models for neurological diseases and concluded that neural differentiation is probably not the leading property of adult MSCs and NCSCs concerning neurological pathology management. A fine analysis of the molecules that are secreted by MSCs and NCSCs would definitely be of significant interest regarding their important contribution to the clinical and pathological recovery after CNS lesions. PMID:23486833

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

PubMed

Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

2009-04-01

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

PubMed

Li, Dong; Zuo, Qisheng; Lian, Chao; Zhang, Lei; Shi, Qingqing; Zhang, Zhentao; Wang, Yingjie; Ahmed, Mahmoud F; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

2015-08-01

We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

In Vitro and in Silico Tools To Assess Extent of Cellular Uptake and Lysosomal Sequestration of Respiratory Drugs in Human Alveolar Macrophages.

PubMed

Ufuk, Ayşe; Assmus, Frauke; Francis, Laura; Plumb, Jonathan; Damian, Valeriu; Gertz, Michael; Houston, J Brian; Galetin, Aleksandra

2017-04-03

Accumulation of respiratory drugs in human alveolar macrophages (AMs) has not been extensively studied in vitro and in silico despite its potential impact on therapeutic efficacy and/or occurrence of phospholipidosis. The current study aims to characterize the accumulation and subcellular distribution of drugs with respiratory indication in human AMs and to develop an in silico mechanistic AM model to predict lysosomal accumulation of investigated drugs. The data set included 9 drugs previously investigated in rat AM cell line NR8383. Cell-to-unbound medium concentration ratio (K p,cell ) of all drugs (5 μM) was determined to assess the magnitude of intracellular accumulation. The extent of lysosomal sequestration in freshly isolated human AMs from multiple donors (n = 5) was investigated for clarithromycin and imipramine (positive control) using an indirect in vitro method (±20 mM ammonium chloride, NH 4 Cl). The AM cell parameters and drug physicochemical data were collated to develop an in silico mechanistic AM model. Three in silico models differing in their description of drug membrane partitioning were evaluated; model (1) relied on octanol-water partitioning of drugs, model (2) used in vitro data to account for this process, and model (3) predicted membrane partitioning by incorporating AM phospholipid fractions. In vitro K p,cell ranged >200-fold for respiratory drugs, with the highest accumulation seen for clarithromycin. A good agreement in K p,cell was observed between human AMs and NR8383 (2.45-fold bias), highlighting NR8383 as a potentially useful in vitro surrogate tool to characterize drug accumulation in AMs. The mean K p,cell of clarithromycin (81, CV = 51%) and imipramine (963, CV = 54%) were reduced in the presence of NH 4 Cl by up to 67% and 81%, respectively, suggesting substantial contribution of lysosomal sequestration and intracellular binding in the accumulation of these drugs in human AMs. The in vitro data showed variability in drug accumulation between individual human AM donors due to possible differences in lysosomal abundance, volume, and phospholipid content, which may have important clinical implications. Consideration of drug-acidic phospholipid interactions significantly improved the performance of the in silico models; use of in vitro K p,cell obtained in the presence of NH 4 Cl as a surrogate for membrane partitioning (model (2)) captured the variability in clarithromycin and imipramine K p,cell observed in vitro and showed the best ability to predict correctly positive and negative lysosomotropic properties. The developed mechanistic AM model represents a useful in silico tool to predict lysosomal and cellular drug concentrations based on drug physicochemical data and system specific properties, with potential application to other cell types.

Acoustic Characterization of a Vessel-on-a-Chip Microfluidic System for Ultrasound-Mediated Drug Delivery.

PubMed

Beekers, Ines; van Rooij, Tom; Verweij, Martin D; Versluis, Michel; de Jong, Nico; Trietsch, Sebastiaan J; Kooiman, Klazina

2018-04-01

Ultrasound in the presence of gas-filled microbubbles can be used to enhance local uptake of drugs and genes. To study the drug delivery potential and its underlying physical and biological mechanisms, an in vitro vessel model should ideally include 3-D cell culture, perfusion flow, and membrane-free soft boundaries. Here, we propose an organ-on-a-chip microfluidic platform to study ultrasound-mediated drug delivery: the OrganoPlate. The acoustic propagation into the OrganoPlate was determined to assess the feasibility of controlled microbubble actuation, which is required to study the microbubble-cell interaction for drug delivery. The pressure field in the OrganoPlate was characterized non-invasively by studying experimentally the well-known response of microbubbles and by simulating the acoustic wave propagation in the system. Microbubble dynamics in the OrganoPlate were recorded with the Brandaris 128 ultrahigh-speed camera (17 million frames/s) and a control experiment was performed in an OptiCell, an in vitro monolayer cell culture chamber that is conventionally used to study ultrasound-mediated drug delivery. When insonified at frequencies between 1 and 2 MHz, microbubbles in the OrganoPlate experienced larger oscillation amplitudes resulting from higher local pressures. Microbubbles responded similarly in both systems when insonified at frequencies between 2 and 4 MHz. Numerical simulations performed with a 3-D finite-element model of ultrasound propagation into the OrganoPlate and the OptiCell showed the same frequency-dependent behavior. The predictable and homogeneous pressure field in the OrganoPlate demonstrates its potential to develop an in vitro 3-D cell culture model, well suited to study ultrasound-mediated drug delivery.

Characterization of CurcuEmulsomes: nanoformulation for enhanced solubility and delivery of curcumin.

PubMed

Ucisik, Mehmet H; Küpcü, Seta; Schuster, Bernhard; Sleytr, Uwe B

2013-12-06

Curcumin is a polyphenolic compound isolated from the rhizomes of the plant Curcuma longa and shows intrinsic anti-cancer properties. Its medical use remains limited due to its extremely low water solubility and bioavailability. Addressing this problem, drug delivery systems accompanied by nanoparticle technology have emerged. The present study introduces a novel nanocarrier system, so-called CurcuEmulsomes, where curcumin is encapsulated inside the solid core of emulsomes. CurcuEmulsomes are spherical solid nanoparticles with an average size of 286 nm and a zeta potential of 37 mV. Encapsulation increases the bioavailability of curcumin by up to 10,000 fold corresponding to a concentration of 0.11 mg/mL. Uptaken by HepG2 human liver carcinoma cell line, CurcuEmulsomes show a significantly prolonged biological activity and demonstrated therapeutic efficacy comparable to free curcumin against HepG2 in vitro - with a delay in response, as assessed by cell viability, apoptosis and cell cycle studies. The delay is attributed to the solid character of the nanocarrier prolonging the release of curcumin inside the HepG2 cells. Incorporation of curcumin into emulsomes results in water-soluble and stable CurcuEmulsome nanoformulations. CurcuEmulsomes do not only successfully facilitate the delivery of curcumin into the cell in vitro, but also enable curcumin to reach its effective concentrations inside the cell. The enhanced solubility of curcumin and the promising in vitro efficacy of CurcuEmulsomes highlight the potential of the system for the delivery of lipophilic drugs. Moreover, high degree of compatibility, prolonged release profile and tailoring properties feature CurcuEmulsomes for further therapeutic applications in vivo.

Characterization of CurcuEmulsomes: nanoformulation for enhanced solubility and delivery of curcumin

PubMed Central

2013-01-01

Background Curcumin is a polyphenolic compound isolated from the rhizomes of the plant Curcuma longa and shows intrinsic anti-cancer properties. Its medical use remains limited due to its extremely low water solubility and bioavailability. Addressing this problem, drug delivery systems accompanied by nanoparticle technology have emerged. The present study introduces a novel nanocarrier system, so-called CurcuEmulsomes, where curcumin is encapsulated inside the solid core of emulsomes. Results CurcuEmulsomes are spherical solid nanoparticles with an average size of 286 nm and a zeta potential of 37 mV. Encapsulation increases the bioavailability of curcumin by up to 10,000 fold corresponding to a concentration of 0.11 mg/mL. Uptaken by HepG2 human liver carcinoma cell line, CurcuEmulsomes show a significantly prolonged biological activity and demonstrated therapeutic efficacy comparable to free curcumin against HepG2 in vitro - with a delay in response, as assessed by cell viability, apoptosis and cell cycle studies. The delay is attributed to the solid character of the nanocarrier prolonging the release of curcumin inside the HepG2 cells. Conclusions Incorporation of curcumin into emulsomes results in water-soluble and stable CurcuEmulsome nanoformulations. CurcuEmulsomes do not only successfully facilitate the delivery of curcumin into the cell in vitro, but also enable curcumin to reach its effective concentrations inside the cell. The enhanced solubility of curcumin and the promising in vitro efficacy of CurcuEmulsomes highlight the potential of the system for the delivery of lipophilic drugs. Moreover, high degree of compatibility, prolonged release profile and tailoring properties feature CurcuEmulsomes for further therapeutic applications in vivo. PMID:24314310

Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

NASA Technical Reports Server (NTRS)

Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

2008-01-01

Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting.

PubMed

Jakobsson, Lars; Franco, Claudio A; Bentley, Katie; Collins, Russell T; Ponsioen, Bas; Aspalter, Irene M; Rosewell, Ian; Busse, Marta; Thurston, Gavin; Medvinsky, Alexander; Schulte-Merker, Stefan; Gerhardt, Holger

2010-10-01

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

From embryonic stem cells to functioning germ cells: science, clinical and ethical perspectives.

PubMed

Kiatpongsan, Sorapop

2007-10-01

Embryonic stem cells have been well recognized as cells having a versatile potential to differentiate into all types of cells in the body including germ cells. There are many research studies focusing on the differentiation processes and protocols to derive various types of somatic cells from embryonic stem cells. However, germ cells have unique differentiation process and developmental pathway compared with somatic cells. Consequently, they will require different differentiation protocols and special culture techniques. More understanding and established in vitro systems for gametogenesis will greatly contribute to further progression of knowledge and technology in germ cell biology, reproductive biology and reproductive medicine. Moreover if oocytes can be efficiently produced in vitro, this will play an important role on progression in nuclear transfer and nuclear reprogramming technology. The present article will provide concise review on past important discoveries, current ongoing studies and future views of this challenging research area. An ethical perspective has also been proposed to give comprehensive summary and viewpoint for future clinical application.

Tissue engineering bioreactor systems for applying physical and electrical stimulations to cells.

PubMed

Jin, GyuHyun; Yang, Gi-Hoon; Kim, GeunHyung

2015-05-01

Bioreactor systems in tissue engineering applications provide various types of stimulation to mimic the tissues in vitro and in vivo. Various bioreactors have been designed to induce high cellular activities, including initial cell attachment, cell growth, and differentiation. Although cell-stimulation processes exert mostly positive effects on cellular responses, in some cases such stimulation can also have a negative effect on cultured cells. In this review, we discuss various types of bioreactor and the positive and negative effects of stimulation (physical, chemical, and electrical) on various cultured cell types. © 2014 Wiley Periodicals, Inc.

Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG-SeSe-PEI Delivery System.

PubMed

Zhang, Yuefeng; Yang, Bin; Liu, Yajie; Qin, Wenjie; Li, Chao; Wang, Lantian; Zheng, Wen; Wu, Yulian

2016-05-01

RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG-SeSe-PEI, was developed and its efficacy evaluated. We prepared three mPEG-SeSe-PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG11.6-SeSe-PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG11.6-SeSe-PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG11.6-SeSe-PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

PubMed

Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

2007-07-30

Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

An insight of in vitro transport of PEGylated non-ionic surfactant vesicles (NSVs) across the intestinal polarized enterocyte monolayers.

PubMed

Primavera, Rosita; Palumbo, Paola; Celia, Christian; Cinque, Benedetta; Carata, Elisabetta; Carafa, Maria; Paolino, Donatella; Cifone, Maria Grazia; Di Marzio, Luisa

2018-06-01

PEGylated non-ionic surfactant-based vesicles (NSVs) are promising drug delivery systems for the local, oral and systemic administrations of therapeutics. The aim of this study was to test the cellular biocompatibility and transport of Nile Red-loaded NSVs (NR-NSVs) across the Caco-2-cell monolayers, which represent an in vitro model of human intestinal epithelium. The NR-NSVs assumed a spherical shape with a mean size of 140 nm, and a narrow size distribution. The NR-NSVs did not modify Caco-2 cell viability, which remained unaltered in vitro up to a concentration of 1 mM. The transport studies demonstrated that the NR-NSVs moved across the Caco-2 monolayers without affecting the transepithelial electrical resistance. These results were supported by flow cytometry analysis, which demonstrated that NR-NSVs were internalized inside the Caco-2 cells. Nanoparticle tracking and Transmission Electron Microscopy (TEM) analysis showed the presence of NR-NSVs in the basolateral side of the Caco-2 monolayers. TEM images also showed that NSVs were transported intact across the Caco-2 monolayers, thus demonstrating a predominant transcytosis mechanism of transport through endocytosis. The NSVs did not affect the integrity of the membrane barrier in vitro, and can potentially be used in clinics to increase the oral bioavailability and delivery of therapeutics. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of 2',3'-didehydro-3'-deoxythymidine in an in vitro hollow-fiber pharmacodynamic model system correlates with results of dose-ranging clinical studies.

PubMed Central

Bilello, J A; Bauer, G; Dudley, M N; Cole, G A; Drusano, G L

1994-01-01

We sought to validate an in vitro system which could predict the minimal effect dose of antiretroviral agents. Mixtures of uninfected CEM cells and CEM cells chronically infected with human immunodeficiency virus (HIV) type 1 MN were exposed to 2',3'-didehydro-3'-deoxythymidine (D4T) in vitro in a hollow-fiber model which simulates the plasma concentration-time profile of D4T in patients. Drug concentration was adjusted to simulate continuous intravenous infusion, or an intravenous bolus administered twice daily. The effect of the dosing regimen was measured with viral infectivity, p24 antigen, and reverse transcriptase or PCR for unintegrated HIV DNA. Dose deescalation studies on a twice-daily dosing schedule predicted a minimum effect dose of 0.5 mg/kg of body weight per day which correlated with the results of a clinical trial. Antiviral effect was demonstrated to be independent of schedule for every 12-h dosing versus continuous infusion. Finally, at or near the minimal effect dose, efficacy appeared to depend on the viral load. The ability of this in vitro pharmacodynamic model to assess the response of HIV-infected cells to different doses and schedules of antiviral agents may be useful in the design of optimal dosing regimens for clinical trials but requires validation with other types of antiretroviral agents. PMID:8092842

Evidence for a dopamine intrinsic direct role in the regulation of the ovary reproductive function: in vitro study on rabbit corpora lutea.

PubMed

Parillo, Francesco; Maranesi, Margherita; Mignini, Fiorenzo; Marinelli, Lisa; Di Stefano, Antonio; Boiti, Cristiano; Zerani, Massimo

2014-01-01

Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.

Biosynthesis of reovirus-specified polypeptides: the reovirus s1 mRNA encodes two primary translation products

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

Jacobs, B.L.; Samuel, C.E.

1985-05-01

Reovirus serotypes 1 (Lang strain) and 3 (Dearing strain) code for a hitherto unrecognized low-molecular-weight polypeptide of Mr approximately 12,000. This polypeptide (p12) was synthesized in vitro in L-cell-free protein synthesizing systems programmed with either reovirus serotype 1 mRNA, reovirus serotype 3 mRNA, or with denatured reovirus genome double-stranded RNA, and in vivo in L-cell cultures infected with either reovirus serotype. Pulse-chase experiments in vivo, and the relative kinetics of synthesis of p12 in vitro, indicate that it is a primary translation product. Fractionation of reovirus mRNAs by velocity sedimentation and translation of separated mRNAs in vitro suggests that p12more » is coded for by the s1 mRNA, which also codes for the previously recognized sigma 1 polypeptide. Synthesis of both p12 and sigma 1 in vitro in L-cell-free protein synthesizing systems programmed with denatured reovirus genome double-stranded RNA also suggests that these two polypeptides can be coded by the same mRNA species. It is proposed that the Mr approximately 12,000 polypeptide encoded by the S1 genome segment be designated sigma 1bNS, and that the polypeptide previously designated sigma 1 be renamed sigma 1a.« less

Neuromuscular junction in a microfluidic device.

PubMed

Park, Hyun Sung; Liu, Su; McDonald, John; Thakor, Nitish; Yang, In Hong

2013-01-01

Malfunctions at the site of neuromuscular junction (NMJ) of post-injuries or diseases are major barriers to recovery of function. The ability to efficiently derive motor neurons (MN) from embryonic stem cells has indicated promise toward the development of new therapies in increasing functional outcomes post injury. Recent advances in micro-technologies have provided advanced culture platforms allowing compartmentalization of sub-cellular components of neurons. In this study, we combined these advances in science and technology to develop a compartmentalized in vitro NMJ model. The developed NMJ system is between mouse embryonic stem cell (mESC)-derived MNs and c2c12 myotubes cultured in a compartmentalized polydimethylsiloxane (PDMS) microfluidic device. While some functional in vitro NMJ systems have been reported, this system would further contribute to research in NMJ-related diseases by providing a system to study the site of action of NMJ aimed at improving promoting better functional recovery.

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.

Improving in vitro to in vivo extrapolation by incorporating toxicokinetic measurements: A case study of lindane-induced neurotoxicity

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

Croom, Edward L.; Shafer, Timothy J.; Evans, Marina V.

Approaches for extrapolating in vitro toxicity testing results for prediction of human in vivo outcomes are needed. The purpose of this case study was to employ in vitro toxicokinetics and PBPK modeling to perform in vitro to in vivo extrapolation (IVIVE) of lindane neurotoxicity. Lindane cell and media concentrations in vitro, together with in vitro concentration-response data for lindane effects on neuronal network firing rates, were compared to in vivo data and model simulations as an exercise in extrapolation for chemical-induced neurotoxicity in rodents and humans. Time- and concentration-dependent lindane dosimetry was determined in primary cultures of rat cortical neuronsmore » in vitro using “faux” (without electrodes) microelectrode arrays (MEAs). In vivo data were derived from literature values, and physiologically based pharmacokinetic (PBPK) modeling was used to extrapolate from rat to human. The previously determined EC{sub 50} for increased firing rates in primary cultures of cortical neurons was 0.6 μg/ml. Media and cell lindane concentrations at the EC{sub 50} were 0.4 μg/ml and 7.1 μg/ml, respectively, and cellular lindane accumulation was time- and concentration-dependent. Rat blood and brain lindane levels during seizures were 1.7–1.9 μg/ml and 5–11 μg/ml, respectively. Brain lindane levels associated with seizures in rats and those predicted for humans (average = 7 μg/ml) by PBPK modeling were very similar to in vitro concentrations detected in cortical cells at the EC{sub 50} dose. PBPK model predictions matched literature data and timing. These findings indicate that in vitro MEA results are predictive of in vivo responses to lindane and demonstrate a successful modeling approach for IVIVE of rat and human neurotoxicity. - Highlights: • In vitro to in vivo extrapolation for lindane neurotoxicity was performed. • Dosimetry of lindane in a micro-electrode array (MEA) test system was assessed. • Cell concentrations at the MEA EC{sub 50} equaled rat brain levels associated with seizure. • PBPK-predicted human brain levels at seizure also equaled EC{sub 50} cell concentrations. • In vitro MEA results are predictive of lindane in vivo dose–response in rats/humans.« less

Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

PubMed

Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

2016-07-07

The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Impact of Polymeric Nanoencapsulation on the Bioavailability of Lutein

NASA Astrophysics Data System (ADS)

Kamil, Alison

Lutein, a fat-soluble xanthophyll, contributes partially to the health benefits from consuming plant foods. Like all dietary carotenoids, lutein has a low bioavailability. In addition to increasing the intake of lutein-rich foods to enhance lutein status, delivery of lutein in polymeric nanoparticles (NP) presents a novel approach to enhancing lutein bioavailability. The overall research objective of this project was to investigate, in rats, the impact of nanoencapsulation using poly(lactic-co-glycolic acid) (PLGA) on the pharmacokinetics of lutein. We also used an in vitro cell culture approach utilizing human epithelial colorectal adenocarcinoma (Caco-2) cells grown in both conventional (CONV) and permeable support (PS) systems to investigate the impact of PLGA-NP on the absorption of lutein in intestinal cells. In chapter one, we compared the efficacy of lutein absorption in vitro using Caco-2 cells grown in both CONV and PS systems. We further examined the role of the micelle, the physiological vehicle for lutein within the small intestine, on its intestinal absorption in vitro compared to an organic solvent, ethanol, which is safe and consumed by humans. The finding from this study demonstrated that the CONV system displayed a larger efficacy of lutein uptake by Caco-2 cells. Further, in the PS system, micelle components appeared to facilitate more effective intestinal secretion of lutein. These findings suggest that lutein uptake by Caco-2 cells is subject to the influence of culturing system (CONV vs. PS) and delivery vehicle (ethanol vs. micelle). Chapter two examined the impact of PLGA-NP in rats on lutein pharmacokinetics in plasma and distribution in selected tissues as compared to free lutein. We also investigated the effect of nanoencapsulation on the absorption of lutein in intestinal cells compared to a more physiological vehicle, the micelle, using the PS method. In addition, we explored the need of additional micelles for the ultimate absorption of lutein loaded in a water soluble NP. The findings of the rat study indicated that, compared to free lutein, PLGA-NP improved the pharmacokinetics (Cmax and AUC) of lutein in the plasma of rats and in general promoted lutein accumulation in mesenteric adipose tissue and spleen but not liver. Yet, compared to micellized lutein, although NP improved the maximal concentration of lutein in the plasma of rats as well as in selected tissues it decreased the cell uptake and secretion of lutein in Caco-2 cells. The negative effect of the NP on cell uptake and secretion was partially remedied by the addition of micelle components. These findings suggest that the delivery of lutein within polymeric NP appears to be a promising approach to improving the bioavailability of lutein in rats. The inconsistent results between the rat and cell culture models warrant further investigations to determine which approach better predicts responses in humans. Further, bile salts and phospholipids, which are necessary to stimulate synthesis and secretion of chylomicrons, appear to facilitate more effective intestinal secretion of PLGA-NP lutein. In summary, with Caco-2 cells cultured in the PS system reliably grown to display phenotypes and functions of enterocytes in the small intestine, this in vitro platform enables the generation of information that is closer to the physiology of the absorptive enterocytes. However, although the CONV system has the physiological attributes of colonic tissue, it appeared to display a greater efficacy of lutein uptake by Caco-2 cells which can provide a rapid preliminary tool for methodology development for nutrient absorption studies. Further, the delivery of lutein in polymeric NP appears to be a promising approach to improve the bioavailability of lutein in vivo but raises issues with regard to the comparability and the predictive value of in vitro models to in vivo responses.

Hetero-cellular prototyping by synchronized multi-material bioprinting for rotary cell culture system.

PubMed

Snyder, Jessica; Son, Ae Rin; Hamid, Qudus; Wu, Honglu; Sun, Wei

2016-01-13

Bottom-up tissue engineering requires methodological progress of biofabrication to capture key design facets of anatomical arrangements across micro, meso and macro-scales. The diffusive mass transfer properties necessary to elicit stability and functionality require hetero-typic contact, cell-to-cell signaling and uniform nutrient diffusion. Bioprinting techniques successfully build mathematically defined porous architecture to diminish resistance to mass transfer. Current limitations of bioprinted cell assemblies include poor micro-scale formability of cell-laden soft gels and asymmetrical macro-scale diffusion through 3D volumes. The objective of this work is to engineer a synchronized multi-material bioprinter (SMMB) system which improves the resolution and expands the capability of existing bioprinting systems by packaging multiple cell types in heterotypic arrays prior to deposition. This unit cell approach to arranging multiple cell-laden solutions is integrated with a motion system to print heterogeneous filaments as tissue engineered scaffolds and nanoliter droplets. The set of SMMB process parameters control the geometric arrangement of the combined flow's internal features and constituent material's volume fractions. SMMB printed hepatocyte-endothelial laden 200 nl droplets are cultured in a rotary cell culture system (RCCS) to study the effect of microgravity on an in vitro model of the human hepatic lobule. RCCS conditioning for 48 h increased hepatocyte cytoplasm diameter 2 μm, increased metabolic rate, and decreased drug half-life. SMMB hetero-cellular models present a 10-fold increase in metabolic rate, compared to SMMB mono-culture models. Improved bioprinting resolution due to process control of cell-laden matrix packaging as well as nanoliter droplet printing capability identify SMMB as a viable technique to improve in vitro model efficacy.

Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

PubMed

Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

2012-01-01

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples. Copyright © 2011 Elsevier Inc. All rights reserved.

Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system.

PubMed

Kim, Ha Ryong; Cho, Han Soo; Shin, Da Young; Chung, Kyu Hyuck

2017-02-01

In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

PubMed

Chue, B; Ferguson, T A; Beaman, K D; Rosenman, S J; Cone, R E; Flood, P M; Green, D R

1989-01-01

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

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

Three-dimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics

NASA Technical Reports Server (NTRS)

Goodwin, T. J.; Prewett, T. L.; Spaulding, G. F.; Becker, J. L.

1997-01-01

The Rotating-Wall Vessel (RWV) is a novel in vitro cell culture system used to successfully culture a cell line derived from a heterologous mixed mullerian tumor cell of the ovary. Although the original tumor was comprised of both epithelial and mesodermal components, long-term culture in conventional flasks established a cell line from this tumor with homogeneous epitheliallike growth characteristics (1). Cells from Passage 36 were seeded into a Rotating-Wall Vessel containing Cytodex-3 microcarrier beads. Scanning electron micrographs of tumor cells cultured for 32 d in the RWV showed the presence of heterogeneous cell populations organized into three-dimensional tissuelike architecture. Immunocytochemical analysis confirmed the cellular heterogeneity, as demonstrated by expression of both epithelial and mesenchymal antigens. Reverse transcription polymerase chain reaction amplification demonstrated the presence of mRNA for cellular oncogenes HER-2/neu, H-ras, K-ras, and tumor suppressor p53. Thus, there are two advantages to propagation of tissue in the RWV culture system:(a) tissue diversification representing populations present in the original tumor, and (b) the three-dimensional freedom to organize tissues morphologically akin to those observed in vivo. These data indicate that the RWV culture system is suitable for generating large quantities of ovarian tumor cells in vitro that are amenable to immunocytochemical, oncogenic, morphologic characteristics demonstrated in vivo.

Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

PubMed

Honda, Yoshitomo; Ding, Xianting; Mussano, Federico; Wiberg, Akira; Ho, Chih-Ming; Nishimura, Ichiro

2013-12-05

Stem cell-based disease modeling presents unique opportunities for mechanistic elucidation and therapeutic targeting. The stable induction of fate-specific differentiation is an essential prerequisite for stem cell-based strategy. Bone morphogenetic protein 2 (BMP-2) initiates receptor-regulated Smad phosphorylation, leading to the osteogenic differentiation of mesenchymal stromal/stem cells (MSC) in vitro; however, it requires supra-physiological concentrations, presenting a bottleneck problem for large-scale drug screening. Here, we report the use of a double-objective feedback system control (FSC) with a differential evolution (DE) algorithm to identify osteogenic cocktails of extrinsic factors. Cocktails containing significantly reduced doses of BMP-2 in combination with physiologically relevant doses of dexamethasone, ascorbic acid, beta-glycerophosphate, heparin, retinoic acid and vitamin D achieved accelerated in vitro mineralization of mouse and human MSC. These results provide insight into constructive approaches of FSC to determine the applicable functional and physiological environment for MSC in disease modeling, drug screening and tissue engineering.

Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control

PubMed Central

Honda, Yoshitomo; Ding, Xianting; Mussano, Federico; Wiberg, Akira; Ho, Chih-ming; Nishimura, Ichiro

2013-01-01

Stem cell-based disease modeling presents unique opportunities for mechanistic elucidation and therapeutic targeting. The stable induction of fate-specific differentiation is an essential prerequisite for stem cell-based strategy. Bone morphogenetic protein 2 (BMP-2) initiates receptor-regulated Smad phosphorylation, leading to the osteogenic differentiation of mesenchymal stromal/stem cells (MSC) in vitro; however, it requires supra-physiological concentrations, presenting a bottleneck problem for large-scale drug screening. Here, we report the use of a double-objective feedback system control (FSC) with a differential evolution (DE) algorithm to identify osteogenic cocktails of extrinsic factors. Cocktails containing significantly reduced doses of BMP-2 in combination with physiologically relevant doses of dexamethasone, ascorbic acid, beta-glycerophosphate, heparin, retinoic acid and vitamin D achieved accelerated in vitro mineralization of mouse and human MSC. These results provide insight into constructive approaches of FSC to determine the applicable functional and physiological environment for MSC in disease modeling, drug screening and tissue engineering. PMID:24305548

In vitro propagation of male germline stem cells from piglets.

PubMed

Zheng, Yi; Tian, Xiue; Zhang, Yaqing; Qin, Jinzhou; An, Junhui; Zeng, Wenxian

2013-07-01

To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells. Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors. The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10 ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population. In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.

Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids.

PubMed

Seiffert, M; Stilgenbauer, S; Döhner, H; Lichter, P

2007-09-01

Accumulation of neoplastic cells in B-cell chronic lymphocytic leukemia (B-CLL) is thought to be due to intrinsic defects in the apoptotic machinery of the leukemic cells or to an altered, survival-stimulating microenvironment in vivo. Despite their long survival in vivo, B-CLL cells undergo rapid spontaneous apoptosis ex vivo. To maintain survival in vitro, we established a coculture system using the human bone marrow-derived stromal cell line HS-5. The microenvironment in these cocultures lead to B-CLL cell survival for at least several months and therefore provided a tool for valid in vitro analysis, mimicking the in vivo situation. Although primary B lymphocytes are notoriously resistant to most gene transfer techniques, we achieved high transfection efficiency and cell viability in this coculture system by using a nucleofection-based strategy. Surprisingly, the introduction of circular plasmid DNA into B cells and B-CLL cells induced rapid apoptosis, which was independent of the type of transgene used, but dependent on the DNA concentration. However, transfection of these cells with mRNA was highly efficient and resulted in sustained cell viability and potent transgene expression. The described procedure represents a new approach to study gene function in primary B cells and B-CLL cells.

Hollow fiber: a biophotonic implant for live cells

NASA Astrophysics Data System (ADS)

Silvestre, Oscar F.; Holton, Mark D.; Summers, Huw D.; Smith, Paul J.; Errington, Rachel J.

2009-02-01

The technical objective of this study has been to design, build and validate biocompatible hollow fiber implants based on fluorescence with integrated biophotonics components to enable in fiber kinetic cell based assays. A human osteosarcoma in vitro cell model fiber system has been established with validation studies to determine in fiber cell growth, cell cycle analysis and organization in normal and drug treated conditions. The rationale for implant development have focused on developing benchmark concepts in standard monolayer tissue culture followed by the development of in vitro hollow fiber designs; encompassing imaging with and without integrated biophotonics. Furthermore the effect of introducing targetable biosensors into the encapsulated tumor implant such as quantum dots for informing new detection readouts and possible implant designs have been evaluated. A preliminary micro/macro imaging approach has been undertaken, that could provide a mean to track distinct morphological changes in cells growing in a 3D matrix within the fiber which affect the light scattering properties of the implant. Parallel engineering studies have showed the influence of the optical properties of the fiber polymer wall in all imaging modes. Taken all together, we show the basic foundation and the opportunities for multi-modal imaging within an in vitro implant format.

An open-access microfluidic model for lung-specific functional studies at an air-liquid interface.

PubMed

Nalayanda, Divya D; Puleo, Christopher; Fulton, William B; Sharpe, Leilani M; Wang, Tza-Huei; Abdullah, Fizan

2009-10-01

In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.

3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

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

Aly, Hussein Hassan; Shimotohno, Kunitada; Hijikata, Makoto

2009-02-06

Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPAR{alpha}) signaling. Furthermore,more » using PPAR{alpha} agonists and antagonists, we also analyzed the effect of PPAR{alpha} signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.« less

Towards Personalized Medicine Mediated by in Vitro Virus-Based Interactome Approaches

PubMed Central

Ohashi, Hiroyuki; Miyamoto-Sato, Etsuko

2014-01-01

We have developed a simple in vitro virus (IVV) selection system based on cell-free co-translation, using a highly stable and efficient mRNA display method. The IVV system is applicable to the high-throughput and comprehensive analysis of proteins and protein–ligand interactions. Huge amounts of genomic sequence data have been generated over the last decade. The accumulated genetic alterations and the interactome networks identified within cells represent a universal feature of a disease, and knowledge of these aspects can help to determine the optimal therapy for the disease. The concept of the “integrome” has been developed as a means of integrating large amounts of data. We have developed an interactome analysis method aimed at providing individually-targeted health care. We also consider future prospects for this system. PMID:24756093

Inhibiting the immunoproteasome exacerbates the pathogenesis of systemic Candida albicans infection in mice

PubMed Central

Mundt, Sarah; Basler, Michael; Buerger, Stefanie; Engler, Harald; Groettrup, Marcus

2016-01-01

Apart from its role in MHC class I antigen processing, the immunoproteasome has recently been implicated in the modulation of T helper cell differentiation under polarizing conditions in vitro and in the pathogenesis of autoimmune diseases in vivo. In this study, we investigated the influence of LMP7 on T helper cell differentiation in response to the fungus Candida albicans. We observed a strong effect of ONX 0914, an LMP7-selective inhibitor of the immunoproteasome, on IFN-γ and IL-17A production by murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated with C. albicans in vitro. Using a murine model of systemic candidiasis, we could confirm reduced generation of IFN-γ- and IL-17A-producing cells in ONX 0914 treated mice in vivo. Interestingly, ONX 0914 treatment resulted in increased susceptibility to systemic candidiasis, which manifested at very early stages of infection. Mice treated with ONX 0914 showed markedly increased kidney and brain fungal burden which resulted in enhanced neutrophil recruitment and immunopathology. Together, these results strongly suggest a role of the immunoproteasome in promoting proinflammatory T helper cells in response to C. albicans but also in affecting the innate antifungal immunity in a T helper cell-independent manner. PMID:26776888

Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

PubMed

Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

2009-05-01

Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

Ultrathin Ceramic Membranes as Scaffolds for Functional Cell Coculture Models on a Biomimetic Scale

PubMed Central

Jud, Corinne; Ahmed, Sher; Müller, Loretta; Kinnear, Calum; Vanhecke, Dimitri; Umehara, Yuki; Frey, Sabine; Liley, Martha; Angeloni, Silvia; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2015-01-01

Abstract Epithelial tissue serves as an interface between biological compartments. Many in vitro epithelial cell models have been developed as an alternative to animal experiments to answer a range of research questions. These in vitro models are grown on permeable two-chamber systems; however, commercially available, polymer-based cell culture inserts are around 10 μm thick. Since the basement membrane found in biological systems is usually less than 1 μm thick, the 10-fold thickness of cell culture inserts is a major limitation in the establishment of realistic models. In this work, an alternative insert, accommodating an ultrathin ceramic membrane with a thickness of only 500 nm (i.e., the Silicon nitride Microporous Permeable Insert [SIMPLI]-well), was produced and used to refine an established human alveolar barrier coculture model by both replacing the conventional inserts with the SIMPLI-well and completing it with endothelial cells. The structural–functional relationship of the model was evaluated, including the translocation of gold nanoparticles across the barrier, revealing a higher translocation if compared to corresponding polyethylene terephthalate (PET) membranes. This study demonstrates the power of the SIMPLI-well system as a scaffold for epithelial tissue cell models on a truly biomimetic scale, allowing construction of more functionally accurate models of human biological barriers. PMID:26713225

Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

NASA Astrophysics Data System (ADS)

Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

1990-09-01

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

A minocycline-releasing PMMA system as a space maintainer for staged bone reconstructions-in vitro antibacterial, cytocompatibility and anti-inflammatory characterization.

PubMed

Silva, Tiago; Grenho, Liliana; Barros, Joana; Silva, José Carlos; Pinto, Rosana V; Matos, Ana; Colaço, Bruno; Fernandes, Maria Helena; Bettencourt, Ana; Gomes, Pedro S

2017-06-06

In the present work, we study the development and biological characterization of a polymethyl methacrylate (PMMA)-based minocycline delivery system, to be used as a space maintainer within craniofacial staged regenerative interventions. The developed delivery systems were characterized regarding solid state characteristics and assayed in vitro for antibacterial and anti-inflammatory activity, and cytocompatibility with human bone cells. A drug release profile allowed for an initial burst release and a more sustained and controlled release over time, with minimum inhibitory concentrations for the assayed and relevant pathogenic bacteria (i.e., Staphylococcus aureus, slime-producer Staphylococcus epidermidis and Escherichia coli) being easily attained in the early time points, and sustained up to 72 h. Furthermore, an improved osteoblastic cell response-with enhancement of cell adhesion and cell proliferation-and increased anti-inflammatory activity were verified in developed systems, compared to a control (non minocycline-loaded PMMA cement). The obtained results converge to support the possible efficacy of the developed PMMA-based minocycline delivery systems for the clinical management of complex craniofacial trauma. Here, biomaterials with space maintenance properties are necessary for the management of staged reconstructive approaches, thus minimizing the risk of peri-operative infections and enhancing the local tissue healing and early stages of regeneration.

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

PubMed

van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

2017-11-25

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

Ribosome display: next-generation display technologies for production of antibodies in vitro.

PubMed

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Cell mechanics and immune system link up to fight infections

NASA Astrophysics Data System (ADS)

Ekpenyong, Andrew; Man, Si Ming; Tourlomousis, Panagiotis; Achouri, Sarra; Cammarota, Eugenia; Hughes, Katherine; Rizzo, Alessandro; Ng, Gilbert; Guck, Jochen; Bryant, Clare

2015-03-01

Infectious diseases, in which pathogens invade and colonize host cells, are responsible for one third of all mortality worldwide. Host cells use special proteins (immunoproteins) and other molecules to fight viral and bacterial invaders. The mechanisms by which immunoproteins enable cells to reduce bacterial loads and survive infections remain unclear. Moreover, during infections, some immunoproteins are known to alter the cytoskeleton, the structure that largely determines cellular mechanical properties. We therefore used an optical stretcher to measure the mechanical properties of primary immune cells (bone marrow derived macrophages) during bacterial infection. We found that macrophages become stiffer upon infection. Remarkably, macrophages lacking the immunoprotein, NLR-C4, lost the stiffening response to infection. This in vitro result correlates with our in vivo data whereby mice lacking NLR-C4 have more lesions and hence increased bacterial distribution and spread. Thus, the immune-protein-dependent increase in cell stiffness in response to bacterial infection (in vitro result) seems to have a functional role in the system level fight against pathogens (in vivo result). We will discuss how this functional link between cell mechanical properties and innate immunity, effected by actin polymerization, reduces the spread of infection.

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

Role of Somatic Testicular Cells during Mouse Spermatogenesis in Three-Dimensional Collagen Gel Culture System

PubMed Central

Khajavi, Noushafarin; Akbari, Mohammad; Abolhassani, Farid; Dehpour, Ahmad Reza; Koruji, Morteza; Habibi Roudkenar, Mehryar

2014-01-01

Objective Spermatogonial stem cells (SSCs) are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional (3D) culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. Materials and Methods In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting (MACS) using GDNF family receptor alpha-1 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 (SCP3)-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 (TTF1) as post meiotic markers. For statistical analysis student t test was performed. Results Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Conclusion Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro. PMID:24518977

Multifunctional Bioreactor System for Human Intestine Tissues

PubMed Central

2017-01-01

The three-dimensional (3D) cultivation of intestinal cells and tissues in dynamic bioreactor systems to represent in vivo intestinal microenvironments is essential for developing regenerative medicine treatments for intestinal diseases. We have previously developed in vitro human intestinal tissue systems using a 3D porous silk scaffold system with intestinal architectures and topographical features for the adhesion, growth, and differentiation of intestinal cells under static culture conditions. In this study, we designed and fabricated a multifunctional bioreactor system that incorporates pre-epithelialized 3D silk scaffolds in a dynamic culture environment for in vitro engineering of human intestine tissues. The bioreactor system allows for control of oxygen levels in perfusion fluids (aerobic simulated intestinal fluid (SIF), microaerobic SIF, and anaerobic SIF), while ensuring control over the mechanical and chemical microenvironments present in native human intestines. The bioreactor system also enables 3D cell culture with spatial separation and cultivation of cocultured epithelial and stromal cells. Preliminary functional analysis of tissues housed in the bioreactor demonstrated that the 3D tissue constructs survived and maintained typical phenotypes of intestinal epithelium, including epithelial tight junction formation, intestinal biomarker expression, microvilli formation, and mucus secretion. The unique combination of a dynamic bioreactor and 3D intestinal constructs offers utility for engineering human intestinal tissues for the study of intestinal diseases and discovery options for new treatments. PMID:29333491

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

PubMed Central

Park, Jung-Sun; Park, Soo-Young; Cho, Hyun-Il; Sohn, Hyun-Jung

2011-01-01

Background Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-γ ELISPOT assay, cytotoxicity assay and tetramer staining. Results DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines. PMID:21860612

S100A8, An Oocyte-Specific Chemokine, Directs the Migration of Ovarian Somatic Cells During Mouse Primordial Follicle Assembly.

PubMed

Teng, Zhen; Wang, Chao; Wang, Yijing; Huang, Kun; Xiang, Xi; Niu, Wanbao; Feng, Lizhao; Zhao, Lihua; Yan, Hao; Zhang, Hua; Xia, Guoliang

2015-12-01

In the mammalian ovaries, the primordial follicle pool determines the reproductive capability over the lifetime of a female. The primordial follicle is composed of two cell members, namely the oocyte and the pre-granulosa cells that encircle the oocyte. However, it is unclear what factors are involved in the reorganization of the two distinct cells into one functional unit. This study was performed to address this issue. Firstly, in an in vitro reconstruction system, dispersed ovarian cells from murine fetal ovaries at 19.0 days post coitum (dpc) reassembled into follicle-like structures, independent of the physical distance between the cells, implying that either oocytes or ovarian somatic cells (OSCs) were motile. We then carried out a series of transwell assay experiments, and determined that it was in fact 19.0 dpc OSCs (as opposed to oocytes), which exhibited a significant chemotactic response to both fetal bovine serum and oocytes themselves. We observed that S100A8, a multi-functional chemokine, may participate in the process as it is mainly expressed in oocytes within the cysts/plasmodia. S100A8 significantly promoted the number of migrating OSCs by 2.5 times in vitro, of which 66.9% were FOXL2 protein-positive cells, implying that the majority of motile OSCs were pre-granulosa cells. In addition, an S100A8-specific antibody inhibited the formation of follicle-like reconstruction cell mass in vitro. And, the primordial follicle formation was reduced when S100a8-specific siRNA was applied onto in vitro cultured 17.5 dpc ovary. Therefore, S100A8 could be a chemokine of oocyte origin, which attracts OSCs to form the primordial follicles. © 2015 Wiley Periodicals, Inc.

Tungsten carbide-cobalt as a nanoparticulate reference positive control in in vitro genotoxicity assays.

PubMed

Moche, Hélène; Chevalier, Dany; Barois, Nicolas; Lorge, Elisabeth; Claude, Nancy; Nesslany, Fabrice

2014-01-01

With the increasing human exposure to nanoparticles (NP), the evaluation of their genotoxic potential is of significant importance. However, relevance for NP of the routinely used in vitro genotoxicity assays is often questioned, and a nanoparticulate reference positive control would therefore constitute an important step to a better testing of NP, ensuring that test systems are really appropriate. In this study, we investigated the possibility of using tungsten carbide-cobalt (WC-Co) NP as reference positive control in in vitro genotoxicity assays, including 2 regulatory assays, the mouse lymphoma assay and the micronucleus assay, and in the Comet assay, recommended for the toxicological evaluation of nanomedicines by the French Agency of Human Health Products (Afssaps). Through these assays, we were able to study different genetic endpoints in 2 cell types commonly used in regulatory genotoxicity assays: the L5178Y mouse lymphoma cell line and primary cultures of human lymphocytes. Our results showed that the use of WC-Co NP as positive control in in vitro genotoxicity assays was conceivable, but that different parameters have to be considered, such as cell type and treatment schedule. L5178Y mouse lymphoma cells did not provide satisfactory results in the 3 performed tests. However, human lymphocytes were more sensitive to genotoxic effects induced by WC-Co NP, particularly after a 24-h treatment in the in vitro micronucleus assay and after a 4-h treatment in the in vitro Comet assay. Under such conditions, WC-Co could be used as a nanoparticulate reference positive control in these assays.

National Cancer Institute Pediatric Preclinical Testing Program: Model Description for In Vitro Cytotoxicity Testing

PubMed Central

Kang, Min H.; Smith, Malcolm A.; Morton, Christopher L.; Keshelava, Nino; Houghton, Peter J.; Reynolds, C. Patrick

2010-01-01

Background The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program (PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials. Procedures In vitro cytotoxicity testing employs a semi-automated fluorescence-based digital imaging cytotoxicity assay (DIMSCAN) that has a 4-log dynamic range of detection. Curve fitting of the fractional survival data of the cell lines in response to various concentrations of the agents was used to calculate relative IC50, absolute IC50, and Ymin values The panel of 23 pediatric cancer cell lines included leukemia (n=6), lymphoma (n=2), rhabdomyosarcoma (n=4), brain tumors (n=3), Ewing family of tumors (EFT, n=4), and neuroblastoma (n=4). The doubling times obtained using DIMSCAN were incorporated into data analyses to estimate the relationship between input cell numbers and final cell number. Results We report in vitro activity data for three drugs (vincristine, melphalan, and etoposide) that are commonly used for pediatric cancer and for the mTOR inhibitor rapamycin, an agent that is currently under preclinical investigation for cancer. To date, the PPTP has completed in vitro testing of 39 investigational and approved agents for single drug activity and two investigational agents in combination with various “standard” chemotherapy drugs. Conclusions This robust in vitro cytotoxicity testing system for pediatric cancers will enable comparisons to response data for novel agents obtained from xenograft studies and from clinical trials. PMID:20922763

Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

PubMed

Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

2009-01-01

Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

PubMed

Futrega, Kathryn; Atkinson, Kerry; Lott, William B; Doran, Michael R

2017-04-01

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25-400 MSCs and 10 umbilical cord blood (CB)-derived CD34 + progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34 + cell expansion outcomes. By contrast, a substantial increase in CD34 + CD38 - cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34 + CD38 - cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34 + CD38 - cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34 + cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.

Decreased nitric oxide levels stimulate osteoclastogenesis and bone resorption both in vitro and in vivo on the chick chorioallantoic membrane in association with neoangiogenesis.

PubMed

Collin-Osdoby, P; Rothe, L; Bekker, S; Anderson, F; Osdoby, P

2000-03-01

High nitric oxide (NO) levels inhibit osteoclast (OC)-mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen-deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC-like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose-dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular-independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC-like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose-dependently inhibited in vitro OC-like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC-mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC-mediated bone remodeling via AG or other pro-angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

PubMed

Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

2016-09-01

Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. None. This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture

PubMed Central

Reda, A.; Hou, M.; Winton, T.R.; Chapin, R.E.; Söder, O.; Stukenborg, J.-B.

2016-01-01

STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. PMID:27430551

Engineered Muscle Actuators: Cells and Tissues

DTIC Science & Technology

2007-01-10

tissue culture perfusion bioreactors The UNC group led the development of the final version of the integrated cell culture bioreactor . The system was...construct engineered in vitro from primary mammalian cells (C) The first demonstration of developmental improvements in engineered tendon constitutive...2007 Final Performance Report 1 Nov 2004 - 31 Oct 2006 4. TITLE AND SUBTITLE 5.. CONTRACT NUMBER Engineered Muscle Actuators: Cells and Tissues FA9550

Improvement of In Vitro Three‐Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor

PubMed Central

Chen, Jie; Yuan, Zhaoyuan; Liu, Yu; Zheng, Rui; Dai, Yao; Tao, Ran; Xia, Huitang; Liu, Hairong; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin

2016-01-01

Abstract In vitro three‐dimensional (3D) cartilage regeneration is a promising strategy for repair of cartilage defects. However, inferior mechanical strength and tissue homogeneity greatly restricted its clinical translation. Simulation of mechanical stress through a bioreactor is an important approach for improving in vitro cartilage regeneration. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure‐transmitting mode achieved by slight deformation of a flexible membrane in a completely sealed stainless steel device. The newly developed bioreactor efficiently avoided the potential risks of previously reported pressure‐transmitting modes and simultaneously addressed a series of important issues, such as pressure scopes, culture chamber sizes, sealability, contamination control, and CO2 balance. The whole bioreactor system realized stable long‐term (8 weeks) culture under high HP (5–10 MPa) without the problems of medium leakage and contamination. Furthermore, the results of in vitro 3D tissue culture based on a cartilage regeneration model revealed that HP provided by the newly developed bioreactor efficiently promoted in vitro 3D cartilage formation by improving its mechanical strength, thickness, and homogeneity. Detailed analysis in cell proliferation, cartilage matrix production, and cross‐linking level of collagen macromolecules, as well as density and alignment of collagen fibers, further revealed the possible mechanisms that HP regulated in vitro cartilage regeneration. The current study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus will help to accelerate its clinical translation. Stem Cells Translational Medicine 2017;6:982–991 PMID:28297584

Characterisation of the borgwaldt LM4E system for in vitro exposures to undiluted aerosols from next generation tobacco and nicotine products (NGPs).

PubMed

Adamson, Jason; Jaunky, Tomasz; Thorne, David; Gaça, Marianna D

2018-03-01

Traditional in vitro exposure to combustible tobacco products utilise exposure systems that include the use of smoking machines to generate, dilute and deliver smoke to in vitro cell cultures. With reported lower emissions from next generation tobacco and nicotine products (NGPs), including e-cigarettes and tobacco heating products (THPs), diluting the aerosol is potentially not required. Herein we present a simplified exposure scenario to undiluted NGP aerosols, using a new puffing system called the LM4E. Nicotine delivery from an e-cigarette was used as a dosimetry marker, and was measured at source across 4 LM4E ports and in the exposure chamber. Cell viability studies, using Neutral Red Uptake (NRU) assay, were performed using H292 human lung epithelial cells, testing undiluted aerosols from an e-cigarette and a THP. E-cigarette mean nicotine generated at source was measured at 0.084 ± 0.005 mg/puff with no significant differences in delivery across the 4 different ports, p = 0.268 (n = 10/port). Mean nicotine delivery from the e-cigarette to the in vitro exposure chamber (measured up to 100 puffs) was 0.046 ± 0.006 mg/puff, p = 0.061. Aerosol penetration within the LM4E was 55% from source to chamber. H292 cells were exposed to undiluted e-cigarette aerosol for 2 h (240 puffs) or undiluted THP aerosol for 1 h (120 puffs). There were positive correlations between puff number and nicotine in the exposed culture media, R 2  = 0.764 for the e-cigarette and R 2  = 0.970 for the THP. NRU determined cell viability for e-cigarettes after 2 h' exposure resulted in 21.5 ± 17.0% cell survival, however for the THP, full cytotoxicity was reached after 1-h exposure. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparative study of in vitro and in vivo drug effects on cell-mediated cytotoxicity.

PubMed Central

Borel, J F

1976-01-01

Cell-mediated cytolysis (CMC) was assayed in a system using spleen cells from mice (C57BL/6) sensitized with allogeneic tumour cells (DBA/2 mastocytoma P-815). Anti-inflammatory drugs, immunosuppressives, inhibitors of cell division and other agents were investigated for their capacity to inhibit CMC in three different ways. First, inhibition of CMC after in vitro addition of drug was observed with corticosteroids, some immunosuppressives and inhibitors of cell division. Secondly, suppression of CMC after a single drug administration to sensitized mice shortly before being killed was found with corticosteroids, several immunosuppressives and irradiation. Thirdly, prevention of development of CMC by repeated drug treatment (immunosuppressive schedule) was achieved with most immunosuppressives and cytostatic drugs. Non-steroidal anti-inflammatory drugs were inactive in these tests. Correlation of effects between the three procedures was very poor and it is suggested that various mechanisms may be involved in the different assays. PMID:824198

In vitro interactions between splenocytes and dansylamide dye-embedded nanoparticles detected by flow cytometry

PubMed Central

Nyland, Jennifer F.; Bai, Jennifer J. K.; Katz, Howard E.; Silbergeld, Ellen K.

2009-01-01

Engineered nanoparticles (NPs) possess a range of biological activity. In vitro methods for assessing toxicity and efficacy would be enhanced by simultaneous quantitative information on the behavior of NPs in culture systems and signals of cell response. We have developed a method for visualizing NPs within cells using standard flow cytometric techniques and uniquely designed spherical siloxane NPs with an embedded (covalently bound) dansylamide dye. This method allowed NP visualization without obscuring detection of relevant biomarkers of cell subtype, activation state, and other events relevant to assessing bioactivity. We determined that NPs penetrated cells and induced a range of biological signals consistent with activation and costimulation. These results indicate that NPs may affect cell function at concentrations below those inducing cytotoxicity or apoptosis and demonstrate a novel method to image both localization of NPs and cell-level effects. PMID:19523425

Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo.

PubMed

Cedeño, Cesyen; La Monaca, Esther; Esposito, Mara; Gutierrez, Gustavo J

2016-01-01

The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells.

A novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da

2009-08-01

The pursuit of efficient and highly targeting-selective transporters is an active topic in cancer-targeting therapy. In this study, a novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes (SWCNTs) was developed to investigate cancer cell targeting in vitro. SWCNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). PL-PEG functionalized SWCNTs were then conjugated with fluorescein isothiocyanate (FITC) labeled integrin αvβ3 monoclonal antibody to construct SWCNT-integrin αvβ3 monoclonal antibody system (denoted as SWCNT-PEG-mAb). In vitro study revealed that the system had a high efficiency in cancer cell targeting in integrin αvβ3 positive U87MG cells. Moreover, the SWCNT-PEG-mAb is stable in physiological media, and can be readily transported into U87MG cells via integrin αvβ3-mediated endocytosis in cell. In summary, the integrin αvβ3 monoclonal antibody labeled SWCNT is a potential carrier-candidate for cancer-imaging and drug-delivering in cancer-targeting therapy.

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.

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

PubMed

Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

2016-11-18

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

GM-CSF Monocyte-Derived Cells and Langerhans Cells As Part of the Dendritic Cell Family

PubMed Central

Lutz, Manfred B.; Strobl, Herbert; Schuler, Gerold; Romani, Nikolaus

2017-01-01

Dendritic cells (DCs) and macrophages (Mph) share many characteristics as components of the innate immune system. The criteria to classify the multitude of subsets within the mononuclear phagocyte system are currently phenotype, ontogeny, transcription patterns, epigenetic adaptations, and function. More recently, ontogenetic, transcriptional, and proteomic research approaches uncovered major developmental differences between Flt3L-dependent conventional DCs as compared with Mphs and monocyte-derived DCs (MoDCs), the latter mainly generated in vitro from murine bone marrow-derived DCs (BM-DCs) or human CD14+ peripheral blood monocytes. Conversely, in vitro GM-CSF-dependent monocyte-derived Mphs largely resemble MoDCs whereas tissue-resident Mphs show a common embryonic origin from yolk sac and fetal liver with Langerhans cells (LCs). The novel ontogenetic findings opened discussions on the terminology of DCs versus Mphs. Here, we bring forward arguments to facilitate definitions of BM-DCs, MoDCs, and LCs. We propose a group model of terminology for all DC subsets that attempts to encompass both ontogeny and function. PMID:29109731

Apoptosis induction and anti-cancer activity of LeciPlex formulations.

PubMed

Dhawan, Vivek V; Joshi, Ganesh V; Jain, Ankitkumar S; Nikam, Yuvraj P; Gude, Rajiv P; Mulherkar, Rita; Nagarsenker, Mangal S

2014-10-01

Cationic agents have been reported to possess anti-neoplastic properties against various cancer cell types. However, their complexes with lipids appear to interact differently with different cancer cells. The purpose of this study was to (i) design and generate novel cationic lecithin nanoparticles, (ii) assess and understand the mechanism underlying their putative cytotoxicity and (iii) test their effect on cell cycle progression in various cancer-derived cell lines. In addition, we aimed to evaluate the in vivo potential of these newly developed nanoparticles in oral anti-cancer delivery. Cationic lecithin nanoparticles were generated using a single step nanoprecipitation method and they were characterized for particle size, zeta potential, stability and in vitro release. Their cytotoxic potential was assessed using a sulforhodamine B assay, and their effect on cell cycle progression was evaluated using flow cytometry. The nanoparticle systems were also tested in vivo for their anti-tumorigenic potential. In contrast to cationic agents alone, the newly developed nanoformulations showed a specific toxicity against cancer cells. The mechanism of toxic cell death included apoptosis, S and G2/M cell cycle phase arrest, depending on the type of cationic agent and the cancer-derived cell line used. Both blank and drug-loaded systems exhibited significant anti-cancer activity, suggesting a synergistic anti-tumorigenic effect of the drug and its delivery system. Both in vitro and in vivo data indicate that cationic agents themselves exhibit broad anti-neoplastic activities. Complex formation of the cationic agents with phospholipids was found to provide specificity to the anti-cancer activity. These formulations thus possess potential for the design of effective anti-cancer delivery systems.

A new method using insert-based systems (IBS) to improve cell behavior study on flexible and rigid biomaterials.

PubMed

Grenade, Charlotte; Moniotte, Nicolas; Rompen, Eric; Vanheusden, Alain; Mainjot, Amélie; De Pauw-Gillet, Marie-Claire

2016-12-01

In vitro studies about biomaterials biological properties are essential screening tests. Yet cell cultures encounter difficulties related to cell retention on material surface or to the observation of both faces of permeable materials. The objective of the present study was to develop a reliable in vitro method to study cell behavior on rigid and flexible/permeable biomaterials elaborating two specific insert-based systems (IBS-R and IBS-F respectively). IBS-R was designed as a specific cylindrical polytetrafluoroethylene (PTFE) system to evaluate attachment, proliferation and morphology of human gingival fibroblasts (HGFs) on grade V titanium and lithium disilicate glass-ceramic discs characteristics of dental prostheses. The number of cells, their covering on discs and their morphology were determined from MTS assays and microscopic fluorescent images after 24, 48 and 72 h. IBS-F was developed as a two components system to study HGFs behavior on guided bone regeneration polyester membranes. The viability and the membrane barrier effect were evaluated by metabolic MTS assays and by scanning electron microscopy. IBS-R and IBS-F were shown to promote (1) easy and rapid handling; (2) cell retention on biomaterial surface; (3) accurate evaluation of the cellular proliferation, spreading and viability; (4) use of non-toxic material. Moreover IBS-F allowed the study of the cell migration through degradable membranes, with an access to both faces of the biomaterial and to the bottom of culture wells for medium changing.

An Incubatable Direct Current Stimulation System for In Vitro Studies of Mammalian Cells

PubMed Central

Panitch, Alyssa; Caplan, Michael; Sweeney, James D.

2012-01-01

Abstract The purpose of this study was to provide a simplified alternative technology and format for direct current stimulation of mammalian cells. An incubatable reusable stimulator was developed that effectively delivers a regulated current and does not require constant monitoring. PMID:23514694

Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix.

PubMed

Amorim, Christiani A; Van Langendonckt, Anne; David, Anu; Dolmans, Marie-Madeleine; Donnez, Jacques

2009-01-01

Ovarian tissue cryopreservation is a promising technique to safeguard fertility in cancer patients. However, in some types of cancer, there is a risk of transmitting malignant cells present in the cryopreserved tissue. To avoid such a risk, pre-antral follicles could be isolated from ovarian tissue and grown in vitro. On the basis of this assumption, the aim of our study was to investigate in vitro survival and growth of pre-antral follicles after cryopreservation of ovarian tissue and follicular isolation, followed by encapsulation in alginate beads. Ovarian biopsies from four patients were frozen and thawed. Pre-antral follicles were then isolated and embedded in an alginate matrix before in vitro culture for 7 days. Small pre-antral follicles (42.98 +/- 9.06 microm) from frozen-thawed tissue can survive and develop after enzymatic isolation and in vitro culture. A total of 159 follicles were incubated in a three-dimensional system (alginate hydrogel) and, after 7 days, all of them showed an increase in size (final size 56.73 +/- 13.10 microm). The survival rate of the follicles was 90% (oocyte and all granulosa cells viable). Our preliminary results indicate that alginate hydrogels may be a suitable system for in vitro culture of isolated human pre-antral follicles. However, more studies are required to establish whether follicular morphology and functionality can be maintained using this matrix.

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

PubMed

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

2016-04-01

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. © 2016 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

In vitro anticancer activity, toxicity and structure-activity relationships of phyllostictine A, a natural oxazatricycloalkenone produced by the fungus Phyllosticta cirsii

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

Le Calve, Benjamin; Lallemand, Benjamin; Perrone, Carmen

2011-07-01

The in vitro anticancer activity and toxicity of phyllostictine A, a novel oxazatricycloalkenone recently isolated from a plant-pathogenic fungus (Phyllosticta cirsii) was characterized in six normal and five cancer cell lines. Phyllostictine A displays in vitro growth-inhibitory activity both in normal and cancer cells without actual bioselectivity, while proliferating cells appear significantly more sensitive to phyllostictine A than non-proliferating ones. The main mechanism of action by which phyllostictine displays cytotoxic effects in cancer cells does not seem to relate to a direct activation of apoptosis. In the same manner, phyllostictine A seems not to bind or bond with DNA asmore » part of its mechanism of action. In contrast, phyllostictine A strongly reacts with GSH, which is a bionucleophile. The experimental data from the present study are in favor of a bonding process between GSH and phyllostictine A to form a complex though Michael attack at C=C bond at the acrylamide-like system. Considering the data obtained, two new hemisynthesized phyllostictine A derivatives together with three other natural phyllostictines (B, C and D) were also tested in vitro in five cancer cell lines. Compared to phyllostictine A, the two derivatives displayed a higher, phyllostictines B and D a lower, and phyllostictine C an almost equal, growth-inhibitory activity, respectively. These results led us to propose preliminary conclusions in terms of the structure-activity relationship (SAR) analyses for the anticancer activity of phyllostictine A and its related compounds, at least in vitro.« less

A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

PubMed

Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

2013-01-01

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Generation of human anti-MUC3 IgG antibodies after in vitro immunization of naive peripheral blood B-lymphocytes.

PubMed

Baritaki, S; Zafiropoulos, A; Georgopoulos, E; Souris, S; Krambovitis, E

2001-04-01

It has been demonstrated that IgG antibodies can be generated to self-antigen peptides as well as against viral antigens by an antigen-specific in vitro immunization system of resting human peripheral B-lymphocytes. Using a synthetic peptide from the consensus variable tandem-repeat region of the MUC3 mucin (TSSITTTGTTSHSTPSP) as the B cell epitope, we immunized blood donor B-lymphocytes in vitro and tested for MUC3-specific antibodies by ELISA. After the primary activation step all antibodies were IgM. At the end of the secondary immunization step we obtained 1.8% (21/1138) of the cultures with IgG-switched antibodies. In a competitive inhibition ELISA using the MUC1, MUC2, MUC3, MUC4 and PIP2 peptides, only one culture (F8.1) gave satisfactory specific inhibition. Using this antibody in fluorometric studies, it stained cells from two colon carcinoma cell lines predominantly in the cytoplasm, whereas those from a breast cancer cell line stained predominantly the cell surface. In a preliminary immunohistological evaluation with formalin-fixed sections, the antibody appeared to moderately stain colon sections, but not breast sections or lymph node. This method of in vitro immunization may be a useful tool in generating IgG antibodies specific to self-antigens and could find applications in tumour targeting and immunotherapy.

Ectodermal fragments from normal frog gastrulae condition substrata to support normal and hybrid mesodermal cell migration in vitro.

PubMed

Nakatsuji, N; Johnson, K E

1984-06-01

Using time-lapse cinemicrography and scanning electron microscopy, we have shown that normal Rana embryos and gastrulating hybrid embryos have extracellular fibrils on the inner surface of the ectodermal layer. These fibrils are absent prior to gastrulation and appear in increasing numbers during gastrulation. They can also be deposited in vitro where they condition substrata in such a way that normal presumptive mesodermal cells placed on them show extensive attachment and unoriented cell movement. These fibrils are also present in some arrested hybrid embryos, but in reduced numbers, or are lacking in other arrested hybrid embryos. Explanted ectodermal fragments from arrested hybrid embryos fail both to condition culture substrata by the deposition of fibrils and to promote cell attachment and translocation. In contrast, ectodermal fragments from normal embryos can condition culture substrata so as to promote moderate cell attachment and, for one particular gamete combination, even cell translocation of presumptive mesodermal cells taken from arrested hybrid embryos. These results provide new evidence to support the hypothesis that extracellular fibrils represent a system that promotes mesodermal cell migration in amphibian embryos. Differences in the fibrillar system in urodele and anuran embryos are discussed in relation to fundamental differences in the mode of mesodermal cell migration in these two classes of Amphibia.

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

PubMed Central

Rathinasamy, Vignesh; Toet, Hayley; McCammick, Erin; O’Connor, Anna; Marks, Nikki J.; Mousley, Angela; Brennan, Gerard P.; Halton, David W.; Spithill, Terry W.; Maule, Aaron G.

2016-01-01

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving ‘molecular toolbox’ for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the “neoblast” stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke. PMID:27622752