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Sample records for 3d culture conditions

  1. An Interdisciplinary Conservation Module for Condition Survey on Cultural Heritages with a 3d Information System

    NASA Astrophysics Data System (ADS)

    Pedelì, C.

    2013-07-01

    In order to make the most of the digital outsourced documents, based on new technologies (e.g.: 3D LASER scanners, photogrammetry, etc.), a new approach was followed and a new ad hoc information system was implemented. The obtained product allow to the final user to reuse and manage the digital documents providing graphic tools and an integrated specific database to manage the entire documentation and conservation process, starting from the condition assessment until the conservation / restoration work. The system is organised on two main modules: Archaeology and Conservation. This paper focus on the features and the advantages of the second one. In particular it is emphasized its logical organisation, the possibility to easily mapping by using a very precise 3D metric platform, to benefit of the integrated relational database which allows to well organise, compare, keep and manage different kind of information at different level. Conservation module can manage along the time the conservation process of a site, monuments, object or excavation and conservation work in progress. An alternative approach called OVO by the author of this paper, force the surveyor to observe and describe the entity decomposing it on functional components, materials and construction techniques. Some integrated tools as the "ICOMOS-ISCS Illustrated glossary … " help the user to describe pathologies with a unified approach and terminology. Also the conservation project phase is strongly supported to envision future intervention and cost. A final section is devoted to record the conservation/restoration work already done or in progress. All information areas of the conservation module are interconnected to each other to allows to the system a complete interchange of graphic and alphanumeric data. The conservation module it self is connected to the archaeological one to create an interdisciplinary daily tool.

  2. Unique characteristics of human mesenchymal stem/progenitor cells (MSC) pre-activated in 3D cultures under different conditions

    PubMed Central

    Ylostalo, Joni H.; Bartosh, Thomas J.; Tiblow, April; Prockop, Darwin J.

    2014-01-01

    Background Human mesenchymal stem cells (MSCs) are being employed in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3D. Here we compared the activation of MSCs in 3D in fetal bovine serum (FBS) containing medium and in multiple xeno-free media formulations. Methods MSC aggregation and sphere formation was studied using hanging drop cultures with medium containing FBS or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with gene expression and protein secretion measurements and with functional studies using macrophages and cancer cells. Results MSCs did not condense into tight spheroids and express a full complement of therapeutic genes in MEMα or several commercial stem-cell media. However, we identified a chemically-defined xeno-free media that when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (PGE2, TSG-6) and anti-cancer molecules (TRAIL, IL-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in an LPS-stimulated macrophage system, and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death. Discussion We demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed here for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications. PMID:25231893

  3. Investigating the neuroglial differentiation effect of neuroblastoma conditioned medium in human endometrial stem cells cultured on 3D nanofibrous scaffold.

    PubMed

    Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Norouzi Javidan, Abbas; Ai, Jafar

    2015-08-01

    Neural tissue engineering is an important area of research in the field of tissue-engineering especially for neurodegenerative disease such as spinal cord injury. The differentiation capacity of human endometrial stem cells (hEnSCs) into neuronal cells has yet to be elucidated. Here, the major aim of the present study was to investigate the differentiation ability of hEnSCs cultured on polylactic acid/chitosan (PLA/CS) nanofibrous scaffold into neuroglial cells in response to conditioned medium of BE(2)-C human neuroblastoma cells and growth factors. Here we investigated the use PLA/CS scaffold as a three dimensional (3D) system that increased neuro-glial cells differentiation. Human EnSCs after three passages were differentiated in neuro-glial like cells under neuroblastoma conditioned medium with FGF2/PDGF-AA on PLA/CS scaffold. By day 18, differentiated cells were analyzed for expression of neuroglial markers by qRT-PCR and immunofluorescence. The results revealed that hEnSCs attach, grow and differentiation on the nanofibrous PLA/CS scaffold. Additionally, our study showed the expression of neural and glial lineage markers such as Nestin, NF-L, MAP2, PDGFRa, CNP, Olig2, MBP, and GFAP in the level of mRNA and MAP2, Tuj-1, and NF-L in the protein level after 18 days. Our results demonstrate that hEnSCs cultured on PLA/CS nanofibrous scaffold have the potential to differentiate in neuronal and glial cells in presence of neuroblastoma conditioned medium on PLA/CS scaffold. The result of this study may have impact in tissue engineering and cells-base therapy of neurodegenerative diseases and have a great potential for wide application. PMID:25611196

  4. Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition

    PubMed Central

    Gomes, Aurélie; Guillaume, Ludivine; Grimes, David Robert; Fehrenbach, Jérôme; Lobjois, Valérie; Ducommun, Bernard

    2016-01-01

    The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems. PMID:27575790

  5. Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition.

    PubMed

    Gomes, Aurélie; Guillaume, Ludivine; Grimes, David Robert; Fehrenbach, Jérôme; Lobjois, Valérie; Ducommun, Bernard

    2016-01-01

    The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems. PMID:27575790

  6. 3D Cell Culture in Alginate Hydrogels

    PubMed Central

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

    2015-01-01

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

  7. Interactions between Mesenchymal Stem Cells, Adipocytes, and Osteoblasts in a 3D Tri-Culture Model of Hyperglycemic Conditions in the Bone Marrow Microenvironment

    PubMed Central

    Rinker, Torri E.; Hammoudi, Taymour M.; Kemp, Melissa L.; Lu, Hang; Temenoff, Johnna S.

    2014-01-01

    Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts in high glucose levels. In contrast, MSCs had no reduction of viability or clonogenicity when cultured with adipocytes in high glucose conditions and adipogenic gene expression indicated that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis. PMID:24463781

  8. Filling gaps in cultural heritage documentation by 3D photography

    NASA Astrophysics Data System (ADS)

    Schuhr, W.; Lee, J. D.

    2015-08-01

    This contribution promotes 3D photography as an important tool to obtain objective object information. Keeping mainly in mind World Heritage documentation as well as Heritage protection, it is another intention of this paper, to stimulate the interest in applications of 3D photography for professionals as well as for amateurs. In addition this is also an activity report of the international CIPA task group 3. The main part of this paper starts with "Digging the treasure of existing international 3D photography". This does not only belong to tangible but also to intangible Cultural Heritage. 3D photography clearly supports the recording, the visualization, the preservation and the restoration of architectural and archaeological objects. Therefore the use of 3D photography in C.H. should increase on an international level. The presented samples in 3D represent a voluminous, almost partly "forgotten treasure" of international archives for 3D photography. The next chapter is on "Promoting new 3D photography in Cultural Heritage". Though 3D photographs are a well-established basic photographic and photogrammetric tool, even suited to provide "near real" documentation, they are still a matter of research and improvement. Beside the use of 3D cameras even single lenses cameras are very much suited for photographic 3D documentation purposes in Cultural Heritage. Currently at the Faculty of Civil Engineering of the University of Applied Sciences Magdeburg-Stendal, low altitude aerial photography is exposed from a maximum height of 13m, using a hand hold carbon telescope rod. The use of this "huge selfie stick" is also an (international) recommendation, to expose high resolution 3D photography of monuments under expedition conditions. In addition to the carbon rod recently a captive balloon and a hexacopter UAV- platform is in use, mainly to take better synoptically (extremely low altitude, ground truth) aerial photography. Additional experiments with respect to "easy

  9. Real-time monitoring of 3D cell culture using a 3D capacitance biosensor.

    PubMed

    Lee, Sun-Mi; Han, Nalae; Lee, Rimi; Choi, In-Hong; Park, Yong-Beom; Shin, Jeon-Soo; Yoo, Kyung-Hwa

    2016-03-15

    Three-dimensional (3D) cell cultures have recently received attention because they represent a more physiologically relevant environment compared to conventional two-dimensional (2D) cell cultures. However, 2D-based imaging techniques or cell sensors are insufficient for real-time monitoring of cellular behavior in 3D cell culture. Here, we report investigations conducted with a 3D capacitance cell sensor consisting of vertically aligned pairs of electrodes. When GFP-expressing human breast cancer cells (GFP-MCF-7) encapsulated in alginate hydrogel were cultured in a 3D cell culture system, cellular activities, such as cell proliferation and apoptosis at different heights, could be monitored non-invasively and in real-time by measuring the change in capacitance with the 3D capacitance sensor. Moreover, we were able to monitor cell migration of human mesenchymal stem cells (hMSCs) with our 3D capacitance sensor. PMID:26386332

  10. Culturing Cells in 3D Ordered Cellular Solids

    NASA Astrophysics Data System (ADS)

    Lin, Keng-Hui; Lin, Wang-Jung; Lin, Jing-Ying

    2011-03-01

    Constructing a well-defined 3D microenvironment for cell growth is a key step for tissue engineering and mechanobiology. We demonstrate high-throughput fabrication of gelatin-based ordered cellular solids with tunable pore size and solid fraction. This process involves generating monodisperse liquid foam with a cross-flow microfluidic device. The monodisperse liquid foam was further processed into open-cell solid foam, which was used as scaffolds for 3D cell culture. Three distinct cell types were cultured under these conditions and displayed appropriate physiological, morphological, and functional characteristics. Epithelial cells formed cyst-like structures and were polarized inside pores, myoblasts adopted a tubular structure and fused into myotubes, and fibroblasts exhibited wide varieties of morphologies depending on their location inside the scaffolds. These ordered cellular solids therefore make possible the study of pore-size effects on cells.

  11. Multizone Paper Platform for 3D Cell Cultures

    PubMed Central

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

    2011-01-01

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

  12. 3D Cell Culture Imaging with Digital Holographic Microscopy

    NASA Astrophysics Data System (ADS)

    Dimiduk, Thomas; Nyberg, Kendra; Almeda, Dariela; Koshelva, Ekaterina; McGorty, Ryan; Kaz, David; Gardel, Emily; Auguste, Debra; Manoharan, Vinothan

    2011-03-01

    Cells in higher organisms naturally exist in a three dimensional (3D) structure, a fact sometimes ignored by in vitro biological research. Confinement to a two dimensional culture imposes significant deviations from the native 3D state. One of the biggest obstacles to wider use of 3D cultures is the difficulty of 3D imaging. The confocal microscope, the dominant 3D imaging instrument, is expensive, bulky, and light-intensive; live cells can be observed for only a short time before they suffer photodamage. We present an alternative 3D imaging techinque, digital holographic microscopy, which can capture 3D information with axial resolution better than 2 μm in a 100 μm deep volume. Capturing a 3D image requires only a single camera exposure with a sub-millisecond laser pulse, allowing us to image cell cultures using five orders of magnitude less light energy than with confocal. This can be done with hardware costing ~ 1000. We use the instrument to image growth of MCF7 breast cancer cells and p. pastoras yeast. We acknowledge support from NSF GRFP.

  13. Microfabricated polymeric vessel mimetics for 3-D cancer cell culture

    PubMed Central

    Jaeger, Ashley A.; Das, Chandan K.; Morgan, Nicole Y.; Pursley, Randall H.; McQueen, Philip G.; Hall, Matthew D.; Pohida, Thomas J.; Gottesman, Michael M.

    2013-01-01

    Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell-cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ∼100um drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. We showed oxygen tension gradients inside the clusters oxygenated by synthetic vessels had a ∼100 µm drop-off to anoxia, which is consistent with in vivo studies. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions. PMID:23911071

  14. Microfluidic 3D cell culture: from tools to tissue models.

    PubMed

    van Duinen, Vincent; Trietsch, Sebastiaan J; Joore, Jos; Vulto, Paul; Hankemeier, Thomas

    2015-12-01

    The transition from 2D to 3D cell culture techniques is an important step in a trend towards better biomimetic tissue models. Microfluidics allows spatial control over fluids in micrometer-sized channels has become a valuable tool to further increase the physiological relevance of 3D cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over of signaling gradients. This paper reviews most important developments in microfluidic 3D culture since 2012. Most efforts were exerted in the field of vasculature, both as a tissue on its own and as part of cancer models. We observe that the focus is shifting from tool building to implementation of specific tissue models. The next big challenge for the field is the full validation of these models and subsequently the implementation of these models in drug development pipelines of the pharmaceutical industry and ultimately in personalized medicine applications. PMID:26094109

  15. Nanomagnetic Levitation 3-D Cultures of Breast and Colorectal Cancers

    PubMed Central

    Bumpers, Harvey L.; Janagama, Dasharatham G.; Manne, Upender; Basson, Marc D.; Katkoori, Venkat

    2014-01-01

    Background Innovative technologies for drug discovery and development, cancer models, stem cell research, tissue engineering, and drug testing in various cell-based platforms require an application similar to the in vivo system. Materials and Methods We developed for the first time nanomagnetically levitated three dimensional (3-D) cultures of breast cancer (BC) and colorectal cancer (CRC) cells using carbon encapsulated cobalt magnetic nanoparticles. BC and CRC xenografts grown in severe combined immunodeficient (SCID) mice were evaluated for N-cadherin and Epidermal growth factor receptor (EGFR) expressions. These phenotypes were compared with 2-D cultures and 3-D cultures grown in a gel matrix. Results The BC and CRC cells grown by magnetic levitation formed microtissues. The levitated cultures had high viability and were maintained in culture for long periods of time. It has been observed that N-cadherin and EGFR activities were highly expressed in the levitated 3-D tumor spheres and xenografts of CRC and BC cells. Conclusions Nanomagnetically levitated 3-D cultures tend to form stable microtissues of BC and CRC and may be more feasible for a range of applications in drug discovery or regenerative medicine. PMID:25617973

  16. An Optically Controlled 3D Cell Culturing System

    PubMed Central

    Ishii, Kelly S.; Hu, Wenqi; Namekar, Swapnil A.; Ohta, Aaron T.

    2012-01-01

    A novel 3D cell culture system was developed and tested. The cell culture device consists of a microfluidic chamber on an optically absorbing substrate. Cells are suspended in a thermoresponsive hydrogel solution, and optical patterns are utilized to heat the solution, producing localized hydrogel formation around cells of interest. The hydrogel traps only the desired cells in place while also serving as a biocompatible scaffold for supporting the cultivation of cells in 3D. This is demonstrated with the trapping of MDCK II and HeLa cells. The light intensity from the optically induced hydrogel formation does not significantly affect cell viability. PMID:22701475

  17. 3D spheroid cultures improve the metabolic gene expression profiles of HepaRG cells

    PubMed Central

    Takahashi, Yu; Hori, Yuji; Yamamoto, Tomohisa; Urashima, Toshiki; Ohara, Yasunori; Tanaka, Hideo

    2015-01-01

    3D (three-dimensional) cultures are considered to be an effective method for toxicological studies; however, little evidence has been reported whether 3D cultures have an impact on hepatocellular physiology regarding lipid or glucose metabolism. In the present study, we conducted physiological characterization of hepatoma cell lines HepG2 and HepaRG cells cultured in 3D conditions using a hanging drop method to verify the effect of culture environment on cellular responses. Apo (Apolipoprotein)B as well as albumin secretion was augmented by 3D cultures. Expression of genes related to not only drug, but also glucose and lipid metabolism were significantly enhanced in 3D cultured HepaRG spheroids. Furthermore, mRNA levels of CYP (cytochrome P450) enzymes following exposure to corresponding inducers increased under the 3D condition. These data suggest that this simple 3D culture system without any special biomaterials can improve liver-specific characteristics including lipid metabolism. Considering that the system enables high-throughput assay, it may become a powerful tool for compound screening concerning hepatocellular responses in order to identify potential drugs. PMID:26182370

  18. Polarimetric 3D integral imaging in photon-starved conditions.

    PubMed

    Carnicer, Artur; Javidi, Bahram

    2015-03-01

    We develop a method for obtaining 3D polarimetric integral images from elemental images recorded in low light illumination conditions. Since photon-counting images are very sparse, calculation of the Stokes parameters and the degree of polarization should be handled carefully. In our approach, polarimetric 3D integral images are generated using the Maximum Likelihood Estimation and subsequently reconstructed by means of a Total Variation Denoising filter. In this way, polarimetric results are comparable to those obtained in conventional illumination conditions. We also show that polarimetric information retrieved from photon starved images can be used in 3D object recognition problems. To the best of our knowledge, this is the first report on 3D polarimetric photon counting integral imaging. PMID:25836861

  19. Exploring Cultural Heritage Resources in a 3d Collaborative Environment

    NASA Astrophysics Data System (ADS)

    Respaldiza, A.; Wachowicz, M.; Vázquez Hoehne, A.

    2012-06-01

    Cultural heritage is a complex and diverse concept, which brings together a wide domain of information. Resources linked to a cultural heritage site may consist of physical artefacts, books, works of art, pictures, historical maps, aerial photographs, archaeological surveys and 3D models. Moreover, all these resources are listed and described by a set of a variety of metadata specifications that allow their online search and consultation on the most basic characteristics of them. Some examples include Norma ISO 19115, Dublin Core, AAT, CDWA, CCO, DACS, MARC, MoReq, MODS, MuseumDat, TGN, SPECTRUM, VRA Core and Z39.50. Gateways are in place to fit in these metadata standards into those used in a SDI (ISO 19115 or INSPIRE), but substantial work still remains to be done for the complete incorporation of cultural heritage information. Therefore, the aim of this paper is to demonstrate how the complexity of cultural heritage resources can be dealt with by a visual exploration of their metadata within a 3D collaborative environment. The 3D collaborative environments are promising tools that represent the new frontier of our capacity of learning, understanding, communicating and transmitting culture.

  20. Alzheimer’s in 3D culture: Challenges and perspectives

    PubMed Central

    D'Avanzo, Carla; Aronson, Jenna; Kim, Young Hye; Choi, Se Hoon; Tanzi, Rudolph E.; Kim, Doo Yeon

    2015-01-01

    Summary Alzheimer’s disease (AD) is the most common cause of dementia, and there is currently no cure. The “β-amyloid cascade hypothesis” of AD is the basis of current understanding of AD pathogenesis and drug discovery. However, no AD models have fully validated this hypothesis. We recently developed a human stem cell culture model of AD by cultivating genetically modified human neural stem cells in a three-dimensional (3D) cell culture system. These cells were able to recapitulate key events of AD pathology including β-amyloid plaques and neurofibrillary tangles. In this review, we will discuss the progress and current limitations of AD mouse models and human stem cell models as well as explore the breakthroughs of 3D cell culture systems. We will also share our perspective on the potential of dish models of neurodegenerative diseases for studying pathogenic cascades and therapeutic drug discovery. PMID:26252541

  1. Alzheimer's in 3D culture: challenges and perspectives.

    PubMed

    D'Avanzo, Carla; Aronson, Jenna; Kim, Young Hye; Choi, Se Hoon; Tanzi, Rudolph E; Kim, Doo Yeon

    2015-10-01

    Alzheimer's disease (AD) is the most common cause of dementia, and there is currently no cure. The "β-amyloid cascade hypothesis" of AD is the basis of current understanding of AD pathogenesis and drug discovery. However, no AD models have fully validated this hypothesis. We recently developed a human stem cell culture model of AD by cultivating genetically modified human neural stem cells in a three-dimensional (3D) cell culture system. These cells were able to recapitulate key events of AD pathology including β-amyloid plaques and neurofibrillary tangles. In this review, we will discuss the progress and current limitations of AD mouse models and human stem cell models as well as explore the breakthroughs of 3D cell culture systems. We will also share our perspective on the potential of dish models of neurodegenerative diseases for studying pathogenic cascades and therapeutic drug discovery. PMID:26252541

  2. Single molecule microscopy in 3D cell cultures and tissues.

    PubMed

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. PMID:25453259

  3. 3D Tissue Culturing: Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations (Adv. Healthcare Mater. 13/2016).

    PubMed

    Hagiwara, Masaya; Kawahara, Tomohiro; Nobata, Rina

    2016-07-01

    An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented by M. Hagiwara and co-workers on page 1566. 3D recognition of a sample structure can be achieved by facilitating multi-directional views using a standard microscope without a laser system. The cubic platform has the potential to promote 3D culture studies, offering easy handling and compatibility with commercial culture plates at a low price tag. PMID:27384934

  4. Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

    SciTech Connect

    Han, Ju; Chang, Hang; Giricz, Orsi; Lee, Genee; Baehner, Frederick; Gray, Joe; Bissell, Mina; Kenny, Paraic; Parvin, Bahram

    2010-02-01

    Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPAR? has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPAR? has been validated through two supporting biological assays.

  5. Surface modified alginate microcapsules for 3D cell culture

    NASA Astrophysics Data System (ADS)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  6. Ideal Positions: 3D Sonography, Medical Visuality, Popular Culture.

    PubMed

    Seiber, Tim

    2016-03-01

    As digital technologies are integrated into medical environments, they continue to transform the experience of contemporary health care. Importantly, medicine is increasingly visual. In the history of sonography, visibility has played an important role in accessing fetal bodies for diagnostic and entertainment purposes. With the advent of three-dimensional (3D) rendering, sonography presents the fetus visually as already a child. The aesthetics of this process and the resulting imagery, made possible in digital networks, discloses important changes in the relationship between technology and biology, reproductive health and political debates, and biotechnology and culture. PMID:26164291

  7. Peptide Hydrogels – Versatile Matrices for 3D Cell Culture in Cancer Medicine

    PubMed Central

    Worthington, Peter; Pochan, Darrin J.; Langhans, Sigrid A.

    2015-01-01

    Traditional two-dimensional (2D) cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D) cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell–cell and cell–material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allow for the development of concentration gradients, including oxygen, metabolites, and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability, and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture is short sequence, self-assembling peptide hydrogels. In addition to the review of recent work toward the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture. PMID:25941663

  8. Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

    PubMed Central

    Sun, Xiuzhi S.; Nguyen, Thu A.

    2013-01-01

    Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing. PMID:23527204

  9. Embedding Knowledge in 3D Data Frameworks in Cultural Heritage

    NASA Astrophysics Data System (ADS)

    Coughenour, C. M.; Vincent, M. L.; de Kramer, M.; Senecal, S.; Fritsch, D.; Flores Gutirrez, M.; Lopez-Menchero Bendicho, V. M.; Ioannides, M.

    2015-08-01

    At present, where 3D modeling and visualisation in cultural heritage are concerned, an object's documentation lacks its interconnected memory provided by multidisciplinary examination and linked data. As the layers of paint, wood, and brick recount a structure's physical properties, the intangible, such as the forms of worship through song, dance, burning incense, and oral traditions, contributes to the greater story of its cultural heritage import. Furthermore, as an object or structure evolves through time, external political, religious, or environmental forces can affect it as well. As tangible and intangible entities associated with the structure transform, its narrative becomes dynamic and difficult to easily record. The Initial Training Network for Digital Cultural Heritage (ITN-DCH), a Marie Curie Actions project under the EU 7th Framework Programme, seeks to challenge this complexity by developing a novel methodology capable of offering such a holistic framework. With the integration of digitisation, conservation, linked data, and retrieval systems for DCH, the nature of investigation and dissemination will be augmented significantly. Examples of utilisating and evaluating this framework will range from a UNESCOWorld Heritage site, the Byzantine church of Panagia Forviotissa Asinou in the Troodos Mountains of Cyprus, to various religious icons and a monument located at the Monastery of Saint Neophytos. The application of this effort to the Asinou church, representing the first case study of the ITN-DCH project, is used as a template example in order to assess the technical challenges involved in the creation of such a framework.

  10. Automated full-3D shape measurement of cultural heritage objects

    NASA Astrophysics Data System (ADS)

    Sitnik, Robert; Karaszewski, Maciej; Zaluski, Wojciech; Bolewicki, Pawel

    2009-07-01

    In this paper a fully automated 3D shape measurement system is presented. It consists of rotary stage for cultural heritage objects placement, vertical linear stage with mounted robot arm (with six degrees of freedom) and structured light measurement set-up mounted to its head. All these manipulation devices are automatically controlled by collision detection and next-best-view calculation modules. The goal of whole system is to automatically (without any user attention) and rapidly (from days and weeks to hours) measure whole object. Measurement head is automatically calibrated by the system and its possible working volume starts from centimeters and ends up to one meter. We present some measurement results with different working scenarios along with discussion about its possible applications.

  11. Diachronic 3d Reconstruction for Lost Cultural Heritage

    NASA Astrophysics Data System (ADS)

    Guidi, G.; Russo, M.

    2011-09-01

    Cultural Heritage artifacts can often be underestimated for their hidden presence in the landscape. Such problem is particularly large in countries like Italy, where the massive amount of "famous" artifacts tends to neglect other presences unless properly exposed, or when the remains are dramatically damaged leaving very few interpretation clues to the visitor. In such cases a virtual presentation of the Cultural Heritage site can be of great help, specially for explaining the evolution of its status, giving sometimes sense to few spare stones. The definition of these digital representations deal with two crucial aspects: on the one hand the possibility of 3D surveying the relics in order to have an accurate geometrical image of the current status of the artifact; on the other hand the presence of historical sources both in form of written text or images, that once properly matched with the current geometrical data, may help to recreate digitally a set of 3D models representing visually the various historical phases (diachronic model), up to the current one. The core of this article is the definition of an integrated methodology that starts from an high-resolution digital survey of the remains of an ancient building and develops a coherent virtual reconstruction from different historical sources, suggesting a scalable method suitable to be re-used for generating a 4D (geometry + time) model of the artifact. This approach has been experimented on the "Basilica di San Giovanni in Conca" in Milan, a very significant example for its complex historic evolution that combines evident historic values with an invisible presence inside the city.

  12. Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations.

    PubMed

    Hagiwara, Masaya; Kawahara, Tomohiro; Nobata, Rina

    2016-07-01

    An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented. The 3D structure of biosamples can be recognized without fluorescence. The cubic platform employs two types of hydrogels that are compatible with conventional culture dishes or well plates, facilitating growth in culture, ease of handling, and viewing at multiple angles. PMID:27128576

  13. Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes.

    PubMed

    Yates, Karen E; Allemann, Florin; Glowacki, Julie

    2005-01-01

    Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS(+3)) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering. PMID:15735900

  14. Functional 3-D cardiac co-culture model using bioactive chitosan nanofiber scaffolds.

    PubMed

    Hussain, Ali; Collins, George; Yip, Derek; Cho, Cheul H

    2013-02-01

    The in vitro generation of a three-dimensional (3-D) myocardial tissue-like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long-term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3-D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long-term cardiac function in the 3-D co-culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non-toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono- and co-cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha-sarcomeric actin (SM-actin) and gap junction protein, Connexin-43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long-term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono-cultures resulted in loss of cardiomyocyte polarity and islands of non-coherent contractions. However

  15. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture.

    PubMed

    Utech, Stefanie; Prodanovic, Radivoje; Mao, Angelo S; Ostafe, Raluca; Mooney, David J; Weitz, David A

    2015-08-01

    Monodisperse alginate microgels (10-50 μm) are created via droplet-based microfluidics by a novel crosslinking procedure. Ionic crosslinking of alginate is induced by release of chelated calcium ions. The process separates droplet formation and gelation reaction enabling excellent control over size and homogeneity under mild reaction conditions. Living mesenchymal stem cells are encapsulated and cultured in the generated 3D microenvironments. PMID:26039892

  16. 3D Microperiodic Hydrogel Scaffolds for Robust Neuronal Cultures

    PubMed Central

    Hanson Shepherd, Jennifer N.; Parker, Sara T.; Shepherd, Robert F.; Gillette, Martha U.; Lewis, Jennifer A.; Nuzzo, Ralph G.

    2011-01-01

    Three-dimensional (3D) microperiodic scaffolds of poly(2-hydroxyethyl methacrylate) (pHEMA) have been fabricated by direct-write assembly of a photopolymerizable hydrogel ink. The ink is initially composed of physically entangled pHEMA chains dissolved in a solution of HEMA monomer, comonomer, photoinitiator and water. Upon printing 3D scaffolds of varying architecture, the ink filaments are exposed to UV light, where they are transformed into an interpenetrating hydrogel network of chemically cross-linked and physically entangled pHEMA chains. These 3D microperiodic scaffolds are rendered growth compliant for primary rat hippocampal neurons by absorption of polylysine. Neuronal cells thrive on these scaffolds, forming differentiated, intricately branched networks. Confocal laser scanning microscopy reveals that both cell distribution and extent of neuronal process alignment depend upon scaffold architecture. This work provides an important step forward in the creation of suitable platforms for in vitro study of sensitive cell types. PMID:21709750

  17. Lensfree diffractive tomography for the imaging of 3D cell cultures

    PubMed Central

    Momey, F.; Berdeu, A.; Bordy, T.; Dinten, J.-M.; Marcel, F. Kermarrec; Picollet-D’hahan, N.; Gidrol, X.; Allier, C.

    2016-01-01

    New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm3 of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup. PMID:27231600

  18. Lensfree diffractive tomography for the imaging of 3D cell cultures.

    PubMed

    Momey, F; Berdeu, A; Bordy, T; Dinten, J-M; Marcel, F Kermarrec; Picollet-D'hahan, N; Gidrol, X; Allier, C

    2016-03-01

    New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm (3) of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup. PMID:27231600

  19. Transcriptional profiles of valvular interstitial cells cultured on tissue culture polystyrene, on 2D hydrogels, or within 3D hydrogels

    PubMed Central

    Mabry, Kelly M.; Payne, Samuel Z.; Anseth, Kristi S.

    2015-01-01

    Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis (Chen and Simmons, 2011) [1]. To better understand and quantify how microenvironmental cues influence VIC phenotype, we compared expression profiles of VICs cultured on/in poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. Here, we present both the raw and processed microarray data from these culture conditions. Interpretation of this data can be found in a research article entitled “Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype” (Mabry et al., 2015) [2]. PMID:26702427

  20. A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

    PubMed Central

    Foty, Ramsey

    2011-01-01

    Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of

  1. Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models

    PubMed Central

    Sung, Kyung Eun; Su, Xiaojing; Berthier, Erwin; Pehlke, Carolyn; Friedl, Andreas; Beebe, David J.

    2013-01-01

    The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models. PMID:24124550

  2. Effects of 3-D microwell culture on growth kinetics and metabolism of human embryonic stem cells

    PubMed Central

    Azarin, Samira M.; Larson, Elise A.; Almodóvar-Cruz, Janice M; de Pablo, Juan J.; Palecek, Sean P.

    2013-01-01

    Human embryonic stem cells (hESCs) hold potential in the field of tissue engineering given their capacity for both limitless self-renewal and differentiation to any adult cell type. However, several limitations, including the ability to expand undifferentiated cells and efficiently direct differentiation at scales needed for commercial cell production, prevent realizing the potential of hESCs in tissue engineering. Numerous studies have illustrated that 3-D culture systems provide microenvironmental cues that affect hESC pluripotency and differentiation fates, but little is known about how 3-D culture affects cell expansion. Here we have used a 3-D microwell array to model the differences in hESC growth kinetics and metabolism in 2-D vs. 3-D cultures. Our results demonstrated that 3-D microwell culture reduced hESC size and proliferative capacity, and impacted cell cycle dynamics, lengthening the G1 phase and shortening the G2/M phase of the cell cycle. However, glucose and lactate metabolism were similar in 2-D and 3-D cultures. Elucidating the effects of 3-D culture on growth and metabolism of hESCs may facilitate efforts for developing integrated, scalable cell expansion and differentiation processes with these cells. PMID:23586789

  3. Hydrogels for 3D mammalian cell culture: a starting guide for laboratory practice.

    PubMed

    Ruedinger, Ferdinand; Lavrentieva, Antonina; Blume, Cornelia; Pepelanova, Iliyana; Scheper, Thomas

    2015-01-01

    Hydrogels have become one of the most popular platforms for three-dimensional (3D) cultivation of mammalian cells. The enormous versatility of hydrogel materials makes it possible to design scaffolds with predefined mechanical properties, as well as with desired biofunctionality. 3D hydrogel constructs have been used for a variety of applications, including tissue engineering of microorgan systems, drug delivery, cytotoxicity testing, and drug screening. Moreover, 3D culture is applied for investigating cellular physiology, stem cell differentiation, and tumor models and for studying interaction mechanisms between the extracellular matrix and cells. In this paper, we review current examples of performance-based hydrogel design for 3D cell culture applications. A major emphasis is placed on a description of how standard analytical protocols and imaging techniques are being adapted to analysis of 3D cell culture in hydrogel systems. PMID:25432676

  4. Bioimpedance monitoring of 3D cell culturing--complementary electrode configurations for enhanced spatial sensitivity.

    PubMed

    Canali, Chiara; Heiskanen, Arto; Muhammad, Haseena Bashir; Høyum, Per; Pettersen, Fred-Johan; Hemmingsen, Mette; Wolff, Anders; Dufva, Martin; Martinsen, Ørjan Grøttem; Emnéus, Jenny

    2015-01-15

    A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance spectroscopic (EIS) characterisation were used to determine the configurations' sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics at the corners of the 3D culture chamber. By combining a number of electrode configurations, complementary spatially distributed information on a large 3D cell culture can be obtained with maximised sensitivity in the entire 3D space. The experimental results show that cell proliferation can be monitored within the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering. PMID:25058941

  5. Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer

    SciTech Connect

    Chitcholtan, Kenny; Asselin, Eric; Parent, Sophie; Sykes, Peter H.; Evans, John J.

    2013-01-01

    Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detected by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E{sub 2} (PGE{sub 2}) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.

  6. Photo-crosslinkable hydrogel-based 3D microfluidic culture device.

    PubMed

    Lee, Youlee; Lee, Jong Min; Bae, Pan-Kee; Chung, Il Yup; Chung, Bong Hyun; Chung, Bong Geun

    2015-04-01

    We developed the photo-crosslinkable hydrogel-based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo-crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular-shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel-based 3D microfluidic device, showing that 53-75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo-crosslinkable hydrogel-based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications. PMID:25641332

  7. Microscale 3D collagen cell culture assays in conventional flat-bottom 384-well plates.

    PubMed

    Leung, Brendan M; Moraes, Christopher; Cavnar, Stephen P; Luker, Kathryn E; Luker, Gary D; Takayama, Shuichi

    2015-04-01

    Three-dimensional (3D) culture systems such as cell-laden hydrogels are superior to standard two-dimensional (2D) monolayer cultures for many drug-screening applications. However, their adoption into high-throughput screening (HTS) has been lagging, in part because of the difficulty of incorporating these culture formats into existing robotic liquid handling and imaging infrastructures. Dispensing cell-laden prepolymer solutions into 2D well plates is a potential solution but typically requires large volumes of reagents to avoid evaporation during polymerization, which (1) increases costs, (2) makes drug penetration variable and (3) complicates imaging. Here we describe a technique to efficiently produce 3D microgels using automated liquid-handling systems and standard, nonpatterned, flat-bottomed, 384-well plates. Sub-millimeter-diameter, cell-laden collagen gels are deposited on the bottom of a ~2.5 mm diameter microwell with no concerns about evaporation or meniscus effects at the edges of wells, using aqueous two-phase system patterning. The microscale cell-laden collagen-gel constructs are readily imaged and readily penetrated by drugs. The cytotoxicity of chemotherapeutics was monitored by bioluminescence and demonstrated that 3D cultures confer chemoresistance as compared with similar 2D cultures. Hence, these data demonstrate the importance of culturing cells in 3D to obtain realistic cellular responses. Overall, this system provides a simple and inexpensive method for integrating 3D culture capability into existing HTS infrastructure. PMID:25510473

  8. Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues

    PubMed Central

    Baker, Brendon M.; Chen, Christopher S.

    2012-01-01

    Summary Much of our understanding of the biological mechanisms that underlie cellular functions, such as migration, differentiation and force-sensing has been garnered from studying cells cultured on two-dimensional (2D) glass or plastic surfaces. However, more recently the cell biology field has come to appreciate the dissimilarity between these flat surfaces and the topographically complex, three-dimensional (3D) extracellular environments in which cells routinely operate in vivo. This has spurred substantial efforts towards the development of in vitro 3D biomimetic environments and has encouraged much cross-disciplinary work among biologists, material scientists and tissue engineers. As we move towards more-physiological culture systems for studying fundamental cellular processes, it is crucial to define exactly which factors are operative in 3D microenvironments. Thus, the focus of this Commentary will be on identifying and describing the fundamental features of 3D cell culture systems that influence cell structure, adhesion, mechanotransduction and signaling in response to soluble factors, which – in turn – regulate overall cellular function in ways that depart dramatically from traditional 2D culture formats. Additionally, we will describe experimental scenarios in which 3D culture is particularly relevant, highlight recent advances in materials engineering for studying cell biology, and discuss examples where studying cells in a 3D context provided insights that would not have been observed in traditional 2D systems. PMID:22797912

  9. On-chip clearing of arrays of 3-D cell cultures and micro-tissues.

    PubMed

    Grist, S M; Nasseri, S S; Poon, T; Roskelley, C; Cheung, K C

    2016-07-01

    Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 μm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. PMID:27493703

  10. A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium.

    PubMed

    Speroni, Lucia; Sweeney, Michael F; Sonnenschein, Carlos; Soto, Ana M

    2016-01-01

    The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression. PMID:26891095

  11. Microfluidic 3D cell culture: potential application for tissue-based bioassays

    PubMed Central

    Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

    2014-01-01

    Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

  12. 3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion

    PubMed Central

    Nguyen-Ngoc, Kim-Vy; Shamir, Eliah R.; Huebner, Robert J.; Beck, Jennifer N.; Cheung, Kevin J.; Ewald, Andrew J.

    2016-01-01

    Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called “organoids,” to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems. PMID:25245692

  13. 3D culture assays of murine mammary branching morphogenesis and epithelial invasion.

    PubMed

    Nguyen-Ngoc, Kim-Vy; Shamir, Eliah R; Huebner, Robert J; Beck, Jennifer N; Cheung, Kevin J; Ewald, Andrew J

    2015-01-01

    Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called "organoids," to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems. PMID:25245692

  14. In vivo biomarker expression patterns are preserved in 3D cultures of Prostate Cancer

    SciTech Connect

    Windus, Louisa C.E.; Kiss, Debra L.; Glover, Tristan; Avery, Vicky M.

    2012-11-15

    Here we report that Prostate Cancer (PCa) cell-lines DU145, PC3, LNCaP and RWPE-1 grown in 3D matrices in contrast to conventional 2D monolayers, display distinct differences in cell morphology, proliferation and expression of important biomarker proteins associated with cancer progression. Consistent with in vivo growth rates, in 3D cultures, all PCa cell-lines were found to proliferate at significantly lower rates in comparison to their 2D counterparts. Moreover, when grown in a 3D matrix, metastatic PC3 cell-lines were found to mimic more precisely protein expression patterns of metastatic tumour formation as found in vivo. In comparison to the prostate epithelial cell-line RWPE-1, metastatic PC3 cell-lines exhibited a down-regulation of E-cadherin and {alpha}6 integrin expression and an up-regulation of N-cadherin, Vimentin and {beta}1 integrin expression and re-expressed non-transcriptionally active AR. In comparison to the non-invasive LNCaP cell-lines, PC3 cells were found to have an up-regulation of chemokine receptor CXCR4, consistent with a metastatic phenotype. In 2D cultures, there was little distinction in protein expression between metastatic, non-invasive and epithelial cells. These results suggest that 3D cultures are more representative of in vivo morphology and may serve as a more biologically relevant model in the drug discovery pipeline. -- Highlights: Black-Right-Pointing-Pointer We developed and optimised 3D culturing techniques for Prostate Cancer cell-lines. Black-Right-Pointing-Pointer We investigated biomarker expression in 2D versus 3D culture techniques. Black-Right-Pointing-Pointer Metastatic PC3 cells re-expressed non-transcriptionally active androgen receptor. Black-Right-Pointing-Pointer Metastatic PCa cell lines retain in vivo-like antigenic profiles in 3D cultures.

  15. Capture and 3D culture of colonic crypts and colonoids in a microarray platform

    PubMed Central

    Wang, Yuli; Ahmad, Asad A.; Shah, Pavak K.; Sims, Christopher E.; Magness, Scott T.; Allbritton, Nancy L.

    2013-01-01

    Crypts are the basic structural and functional units of colonic epithelium and can be isolated from the colon and cultured in vitro into multi-cell spheroids termed “colonoids”. Both crypts and colonoids are ideal building blocks for construction of an in vitro tissue model of the colon. Here we proposed and tested a microengineered platform for capture and in vitro 3D culture of colonic crypts and colonoids. An integrated platform was fabricated from polydimethylsiloxane which contained two fluidic layers separated by an array of cylindrical microwells (150-μm diameter, 150-μm depth) with perforated bottoms (30-μm opening, 10-μm depth) termed “microstrainers”. As fluid moved through the array, crypts or colonoids were retained in the microstrainers with a >90% array-filling efficiency. Matrigel as an extracellular matrix was then applied to the microstrainers to generate isolated Matrigel pockets encapsulating the crypts or colonoids. After supplying the essential growth factors, epidermal growth factor, Wnt-3A, R-spondin 2 and noggin, 63±13% of the crypts and 77±8% of the colonoids cultured in the microstrainers over a 48–72 h period formed viable 3D colonoids. Thus colonoid growth on the array was similar to that under standard culture conditions (78±5%). Additionally the colonoids displayed the same morphology and similar numbers of stem and progenitor cells as those under standard culture conditions. Immunofluorescence staining confirmed that the differentiated cell-types of the colon, goblet cells, enteroendocrine cells and absorptive enterocytes, formed on the array. To demonstrating the utility of the array in tracking the colonoid fate, quantitative fluorescence analysis was performed on the arrayed colonoids exposed to reagents such as Wnt-3A and the γ-secretase inhibitor LY-411575. The successful formation of viable, multi-cell type colonic tissue on the microengineered platform represents a first step in the building of a

  16. Microscale 3-D collagen cell culture assays in conventional flat-bottom 384-well plates

    PubMed Central

    Leung, Brendan M.; Moraes, Christopher; Cavnar, Stephen; Luker, Kathryn E.; Luker, Gary D.; Takayama, Shuichi

    2015-01-01

    Three-dimensional culture systems such as cell-laden hydrogels are superior to standard 2-D monolayer cultures for many drug-screening applications. However, their adoption in high throughput screening (HTS) have been lagging, in part due to the difficulty of incorporating these culture formats into existing robotic liquid handling and imaging infrastructures. Dispensing cell-laden pre-polymer solutions into 2-D well-plates is a potential solution, but typically requires large volumes of reagents to avoid evaporation during polymerization, which increases cost, makes drug penetration variable and imaging complex. Here we describe a technique to efficiently produce 3-D ‘microgels’ using automated liquid handling systems and standard, non-patterned, flat-bottomed, 384-well plates. Sub-millimeter-diameter, cell-laden collagen gels are deposited on the bottom of ~2.5 mm-diameter microwell with no concerns over evaporation and meniscus effects at the edges of wells, using aqueous two-phase system patterning. The microscale cell-laden collagen-gel constructs are readily imaged and readily penetrated by drugs. Cytotoxicity of chemotherapeutics were monitored by bioluminescence and demonstrates that 3-D cultures confer chemoresistance, as compared to similar 2-D culture. This data hence, demonstrates the importance of culturing cells in 3-D to obtain realistic cellular responses. Overall, this system provided a simple and inexpensive method for integrating 3-D culture capability into existing HTS infrastructure. PMID:25510473

  17. Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

    PubMed

    Simon, Karen A; Warren, Nicholas J; Mosadegh, Bobak; Mohammady, Marym R; Whitesides, George M; Armes, Steven P

    2015-12-14

    It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel). PMID:26509930

  18. 3D Modeling from Multi-views Images for Cultural Heritage in Wat-Pho, Thailand

    NASA Astrophysics Data System (ADS)

    Soontranon, N.; Srestasathiern, P.; Lawawirojwong, S.

    2015-08-01

    In Thailand, there are several types of (tangible) cultural heritages. This work focuses on 3D modeling of the heritage objects from multi-views images. The images are acquired by using a DSLR camera which costs around 1,500 (camera and lens). Comparing with a 3D laser scanner, the camera is cheaper and lighter than the 3D scanner. Hence, the camera is available for public users and convenient for accessing narrow areas. The acquired images consist of various sculptures and architectures in Wat-Pho which is a Buddhist temple located behind the Grand Palace (Bangkok, Thailand). Wat-Pho is known as temple of the reclining Buddha and the birthplace of traditional Thai massage. To compute the 3D models, a diagram is separated into following steps; Data acquisition, Image matching, Image calibration and orientation, Dense matching and Point cloud processing. For the initial work, small heritages less than 3 meters height are considered for the experimental results. A set of multi-views images of an interested object is used as input data for 3D modeling. In our experiments, 3D models are obtained from MICMAC (open source) software developed by IGN, France. The output of 3D models will be represented by using standard formats of 3D point clouds and triangulated surfaces such as .ply, .off, .obj, etc. To compute for the efficient 3D models, post-processing techniques are required for the final results e.g. noise reduction, surface simplification and reconstruction. The reconstructed 3D models can be provided for public access such as website, DVD, printed materials. The high accurate 3D models can also be used as reference data of the heritage objects that must be restored due to deterioration of a lifetime, natural disasters, etc.

  19. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM.

    PubMed

    Chennell, George; Willows, Robin J W; Warren, Sean C; Carling, David; French, Paul M W; Dunsby, Chris; Sardini, Alessandro

    2016-01-01

    We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. PMID:27548185

  20. Using a 3D Culture System to Differentiate Visceral Adipocytes In Vitro.

    PubMed

    Emont, Margo P; Yu, Hui; Jun, Heejin; Hong, Xiaowei; Maganti, Nenita; Stegemann, Jan P; Wu, Jun

    2015-12-01

    It has long been recognized that body fat distribution and regional adiposity play a major role in the control of metabolic homeostasis. However, the ability to study and compare the cell autonomous regulation and response of adipocytes from different fat depots has been hampered by the difficulty of inducing preadipocytes isolated from the visceral depot to differentiate into mature adipocytes in culture. Here, we present an easily created 3-dimensional (3D) culture system that can be used to differentiate preadipocytes from the visceral depot as robustly as those from the sc depot. The cells differentiated in these 3D collagen gels are mature adipocytes that retain depot-specific characteristics, as determined by imaging, gene expression, and functional assays. This 3D culture system therefore allows for study of the development and function of adipocytes from both depots in vitro and may ultimately lead to a greater understanding of site-specific functional differences of adipose tissues to metabolic dysregulation. PMID:26425808

  1. High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures.

    PubMed

    Joshi, Pranav; Lee, Moo-Yeal

    2015-12-01

    High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology. PMID:26694477

  2. High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures

    PubMed Central

    Joshi, Pranav; Lee, Moo-Yeal

    2015-01-01

    High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology. PMID:26694477

  3. User-Appropriate Viewer for High Resolution Interactive Engagement with 3d Digital Cultural Artefacts

    NASA Astrophysics Data System (ADS)

    Gillespie, D.; La Pensée, A.; Cooper, M.

    2013-07-01

    Three dimensional (3D) laser scanning is an important documentation technique for cultural heritage. This technology has been adopted from the engineering and aeronautical industry and is an invaluable tool for the documentation of objects within museum collections (La Pensée, 2008). The datasets created via close range laser scanning are extremely accurate and the created 3D dataset allows for a more detailed analysis in comparison to other documentation technologies such as photography. The dataset can be used for a range of different applications including: documentation; archiving; surface monitoring; replication; gallery interactives; educational sessions; conservation and visualization. However, the novel nature of a 3D dataset is presenting a rather unique challenge with respect to its sharing and dissemination. This is in part due to the need for specialised 3D software and a supported graphics card to display high resolution 3D models. This can be detrimental to one of the main goals of cultural institutions, which is to share knowledge and enable activities such as research, education and entertainment. This has limited the presentation of 3D models of cultural heritage objects to mainly either images or videos. Yet with recent developments in computer graphics, increased internet speed and emerging technologies such as Adobe's Stage 3D (Adobe, 2013) and WebGL (Khronos, 2013), it is now possible to share a dataset directly within a webpage. This allows website visitors to interact with the 3D dataset allowing them to explore every angle of the object, gaining an insight into its shape and nature. This can be very important considering that it is difficult to offer the same level of understanding of the object through the use of traditional mediums such as photographs and videos. Yet this presents a range of problems: this is a very novel experience and very few people have engaged with 3D objects outside of 3D software packages or games. This paper

  4. Application of 3D hydrodynamic and particle tracking models for better environmental management of finfish culture

    NASA Astrophysics Data System (ADS)

    Moreno Navas, Juan; Telfer, Trevor C.; Ross, Lindsay G.

    2011-04-01

    Hydrographic conditions, and particularly current speeds, have a strong influence on the management of fish cage culture. These hydrodynamic conditions can be used to predict particle movement within the water column and the results used to optimise environmental conditions for effective site selection, setting of environmental quality standards, waste dispersion, and potential disease transfer. To this end, a 3D hydrodynamic model, MOHID, has been coupled to a particle tracking model to study the effects of mean current speed, quiescent water periods and bulk water circulation in Mulroy Bay, Co. Donegal Ireland, an Irish fjard (shallow fjordic system) important to the aquaculture industry. A Lagangrian method simulated the instantaneous release of "particles" emulating discharge from finfish cages to show the behaviour of waste in terms of water circulation and water exchange. The 3D spatial models were used to identify areas of mixed and stratified water using a version of the Simpson-Hunter criteria, and to use this in conjunction with models of current flow for appropriate site selection for salmon aquaculture. The modelled outcomes for stratification were in good agreement with the direct measurements of water column stratification based on observed density profiles. Calculations of the Simpson-Hunter tidal parameter indicated that most of Mulroy Bay was potentially stratified with a well mixed region over the shallow channels where the water is faster flowing. The fjard was characterised by areas of both very low and high mean current speeds, with some areas having long periods of quiescent water. The residual current and the particle tracking animations created through the models revealed an anticlockwise eddy that may influence waste dispersion and potential for disease transfer, among salmon cages and which ensures that the retention time of waste substances from cages is extended. The hydrodynamic model results were incorporated into the ArcView TM GIS

  5. Protein-Engineered Hydrogel Encapsulation for 3-D Culture of Murine Cochlea

    PubMed Central

    Chang, David T.; Chai, Renjie; DiMarco, Rebecca; Heilshorn, Sarah C.; Cheng, Alan G.

    2016-01-01

    Hypothesis Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture. Background Whole-organ culture of the cochlea is a useful model system facilitating manipulation and analysis of live sensory cells and surrounding nonsensory cells. The precisely organized 3-D cochlear structure demands a culture method that preserves this delicate architecture; however, current methods have not been optimized to serve such a purpose. Methods A protein-engineered ELP hydrogel was used to encapsulate organ of Corti isolated from neonatal mice. Cultured cochleae were immunostained for markers of hair cells and supporting cells. Organ of Corti hair cell and supporting cell density and organ dimensions were compared between the ELP and nonencapsulated systems. These culture systems were then compared with noncultured cochlea. Results After 3 days in vitro, vital dye uptake and immunostaining for sensory and nonsensory cells show that encapsulated cochlea contain viable cells with an organized architecture. In comparison with nonencapsulated cultured cochlea, ELP-encapsulated cochleae exhibit higher densities of hair cells and supporting cells and taller and narrower organ of Corti dimensions that more closely resemble those of noncultured cochleae. However, we found compromised cell viability when the culture period extended beyond 3 days. Conclusion We conclude that the ELP hydrogel can help preserve the 3-D architecture of neonatal cochlea in short-term culture, which may be applicable to in vitro study of the physiology and pathophysiology of the inner ear. PMID:25111520

  6. Development of 3D hydrogel culture systems with on-demand cell separation.

    PubMed

    Hamilton, Sharon K; Bloodworth, Nathaniel C; Massad, Christopher S; Hammoudi, Taymour M; Suri, Shalu; Yang, Peter J; Lu, Hang; Temenoff, Johnna S

    2013-04-01

    Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations. PMID:23447378

  7. Hot embossing for fabrication of a microfluidic 3D cell culture platform

    PubMed Central

    Jeon, Jessie S.; Chung, Seok; Kamm, Roger D.; Charest, Joseph L.

    2011-01-01

    Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfludic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact. PMID:21113663

  8. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models.

    PubMed

    Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

    2016-01-01

    BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

  9. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models

    PubMed Central

    Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

    2016-01-01

    BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

  10. Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

    PubMed Central

    Gagliano, Nicoletta; Celesti, Giuseppe; Tacchini, Lorenza; Pluchino, Stefano; Sforza, Chiarella; Rasile, Marco; Valerio, Vincenza; Laghi, Luigi; Conte, Vincenzo; Procacci, Patrizia

    2016-01-01

    AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids. PMID:27182158

  11. Dynamic 3D micropatterned cell co-cultures within photocurable and chemically degradable hydrogels

    PubMed Central

    Sugiura, Shinji; Cha, Jae Min; Yanagawa, Fumiki; Zorlutuna, Pinar; Bae, Hojae; Khademhosseini, Ali

    2014-01-01

    In this paper we report on the development of dynamically controlled 3D micropatterned cellular co-cultures within photocurable and chemically degradable hydrogels. Specifically, we generated dynamic co-cultures of micropatterned murine embryonic stem (mES) cells with human hepatocellular carcinoma (HepG2) cells within 3D hydrogels. HepG2 cells were used due to their ability to direct the differentiation of mES cells through secreted paracrine factors. To generate dynamic co-cultures, mES cells were first encapsulated within micropatterned photocurable poly(ethylene glycol) (PEG) hydrogels. These micropatterned cell-laden PEG hydrogels were subsequently surrounded by calcium alginate (Ca-Alg) hydrogels containing HepG2 cells. After 4 days, the co-culture step was halted by exposing the system to sodium citrate solution, which removed the alginate gels and the encapsulated HepG2 cells. The encapsulated mES cells were then maintained in the resulting cultures for 16 days and cardiac differentiation was analyzed. We observed that the mES cells that were exposed to HepG2 cells in the co-cultures, generated cells with higher expression of cardiac genes and proteins as well as increased spontaneous beating. Due to its ability to control the 3D microenvironment of cells in a spatially and temporally regulated manner the method presented in this study is useful for a range of cell culture applications related to tissue engineering and regenerative medicine. PMID:24170301

  12. 5D Modelling: An Efficient Approach for Creating Spatiotemporal Predictive 3D Maps of Large-Scale Cultural Resources

    NASA Astrophysics Data System (ADS)

    Doulamis, A.; Doulamis, N.; Ioannidis, C.; Chrysouli, C.; Grammalidis, N.; Dimitropoulos, K.; Potsiou, C.; Stathopoulou, E.-K.; Ioannides, M.

    2015-08-01

    Outdoor large-scale cultural sites are mostly sensitive to environmental, natural and human made factors, implying an imminent need for a spatio-temporal assessment to identify regions of potential cultural interest (material degradation, structuring, conservation). On the other hand, in Cultural Heritage research quite different actors are involved (archaeologists, curators, conservators, simple users) each of diverse needs. All these statements advocate that a 5D modelling (3D geometry plus time plus levels of details) is ideally required for preservation and assessment of outdoor large scale cultural sites, which is currently implemented as a simple aggregation of 3D digital models at different time and levels of details. The main bottleneck of such an approach is its complexity, making 5D modelling impossible to be validated in real life conditions. In this paper, a cost effective and affordable framework for 5D modelling is proposed based on a spatial-temporal dependent aggregation of 3D digital models, by incorporating a predictive assessment procedure to indicate which regions (surfaces) of an object should be reconstructed at higher levels of details at next time instances and which at lower ones. In this way, dynamic change history maps are created, indicating spatial probabilities of regions needed further 3D modelling at forthcoming instances. Using these maps, predictive assessment can be made, that is, to localize surfaces within the objects where a high accuracy reconstruction process needs to be activated at the forthcoming time instances. The proposed 5D Digital Cultural Heritage Model (5D-DCHM) is implemented using open interoperable standards based on the CityGML framework, which also allows the description of additional semantic metadata information. Visualization aspects are also supported to allow easy manipulation, interaction and representation of the 5D-DCHM geometry and the respective semantic information. The open source 3DCity

  13. Optimization of a 3D Dynamic Culturing System for In Vitro Modeling of Frontotemporal Neurodegeneration-Relevant Pathologic Features.

    PubMed

    Tunesi, Marta; Fusco, Federica; Fiordaliso, Fabio; Corbelli, Alessandro; Biella, Gloria; Raimondi, Manuela T

    2016-01-01

    Frontotemporal lobar degeneration (FTLD) is a severe neurodegenerative disorder that is diagnosed with increasing frequency in clinical setting. Currently, no therapy is available and in addition the molecular basis of the disease are far from being elucidated. Consequently, it is of pivotal importance to develop reliable and cost-effective in vitro models for basic research purposes and drug screening. To this respect, recent results in the field of Alzheimer's disease have suggested that a tridimensional (3D) environment is an added value to better model key pathologic features of the disease. Here, we have tried to add complexity to the 3D cell culturing concept by using a microfluidic bioreactor, where cells are cultured under a continuous flow of medium, thus mimicking the interstitial fluid movement that actually perfuses the body tissues, including the brain. We have implemented this model using a neuronal-like cell line (SH-SY5Y), a widely exploited cell model for neurodegenerative disorders that shows some basic features relevant for FTLD modeling, such as the release of the FTLD-related protein progranulin (PRGN) in specific vesicles (exosomes). We have efficiently seeded the cells on 3D scaffolds, optimized a disease-relevant oxidative stress experiment (by targeting mitochondrial function that is one of the possible FTLD-involved pathological mechanisms) and evaluated cell metabolic activity in dynamic culture in comparison to static conditions, finding that SH-SY5Y cells cultured in 3D scaffold are susceptible to the oxidative damage triggered by a mitochondrial-targeting toxin (6-OHDA) and that the same cells cultured in dynamic conditions kept their basic capacity to secrete PRGN in exosomes once recovered from the bioreactor and plated in standard 2D conditions. We think that a further improvement of our microfluidic system may help in providing a full device where assessing basic FTLD-related features (including PRGN dynamic secretion) that may be

  14. Additively Manufactured Device for Dynamic Culture of Large Arrays of 3D Tissue Engineered Constructs.

    PubMed

    Costa, Pedro F; Hutmacher, Dietmar W; Theodoropoulos, Christina; Gomes, Manuela E; Reis, Rui L; Vaquette, Cédryck

    2015-04-22

    The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine. PMID:25721231

  15. Polymer-Based Mesh as Supports for Multi-layered 3D Cell Culture and Assays

    PubMed Central

    Simon, Karen A.; Park, Kyeng Min; Mosadegh, Bobak; Subramaniam, Anand Bala; Mazzeo, Aaron; Ngo, Phil M.; Whitesides, George M.

    2013-01-01

    Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system – Cells-in-Gels-in-Mesh (CiGiM) – that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells—layer-by-layer—within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis—(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format. PMID:24095253

  16. Biologic variability of human foreskin fibroblasts in 2D and 3D culture: implications for a wound healing model

    PubMed Central

    2009-01-01

    Background The fibroblast-populated 3D collagen matrix is a model of tissue and healing which has been used since the 1980's. It was hypothesized that anchorage disruption of the collagen matrix would produce p53-dependent apoptosis in the embedded fibroblasts, but results of hypothesis testing were variant. Findings The response of p53 to anchorage disruption in 3D culture or to UV irradiation in 2D culture was influenced both by fibroblast strain and culture conditions. It also was determined that data scatter in a collagen matrix contraction assay was related to fibroblast strain and possibly to technical factors, such as cell culture technician and/or number of matrices utilized. Subsequent analysis suggested that phenotypic drift and/or inter-strain genetic variability may have been responsible for the data scatter. In addition, several technical factors were identified that may have contributed to the scatter. Conclusion Experimentation with human foreskin fibroblasts in both 2D and 3D culture can produce variant data. The underlying cause of the data scatter appears to be partially due to the biologic variability of the fibroblast. PMID:19922655

  17. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    PubMed Central

    Liebmann, Thomas; Rydholm, Susanna; Akpe, Victor; Brismar, Hjalmar

    2007-01-01

    Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. Results Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. Conclusion Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology. PMID:18070345

  18. 3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

    PubMed

    Malenovská, Hana

    2016-02-01

    Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses. PMID:26562056

  19. A 3D human neural cell culture system for modeling Alzheimer’s disease

    PubMed Central

    Kim, Young Hye; Choi, Se Hoon; D’Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J.; Klee, Justin B.; Brüstle, Oliver; Tanzi, Rudolph E.; Kim, Doo Yeon

    2015-01-01

    Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer’s disease (AD) because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel three-dimensional (3D) culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of β-amyloid and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix, and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 days. The aggregation of β-amyloid is observed after 6-weeks of differentiation followed by robust tau pathology after 10–14 weeks. PMID:26068894

  20. A 3D human neural cell culture system for modeling Alzheimer's disease.

    PubMed

    Kim, Young Hye; Choi, Se Hoon; D'Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J; Klee, Justin B; Brüstle, Oliver; Tanzi, Rudolph E; Kim, Doo Yeon

    2015-07-01

    Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer's disease (AD), because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel 3D culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of amyloid-β (Aβ) and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix and the analysis of AD pathogenesis. The 3D culture generation takes 1-2 d. The aggregation of Aβ is observed after 6 weeks of differentiation, followed by robust tau pathology after 10-14 weeks. PMID:26068894

  1. Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

    PubMed Central

    Fai, Stephen; Bennett, Steffany A.L.

    2010-01-01

    The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This

  2. DREAM-3D and the importance of model inputs and boundary conditions

    NASA Astrophysics Data System (ADS)

    Friedel, Reiner; Tu, Weichao; Cunningham, Gregory; Jorgensen, Anders; Chen, Yue

    2015-04-01

    Recent work on radiation belt 3D diffusion codes such as the Los Alamos "DREAM-3D" code have demonstrated the ability of such codes to reproduce realistic magnetospheric storm events in the relativistic electron dynamics - as long as sufficient "event-oriented" boundary conditions and code inputs such as wave powers, low energy boundary conditions, background plasma densities, and last closed drift shell (outer boundary) are available. In this talk we will argue that the main limiting factor in our modeling ability is no longer our inability to represent key physical processes that govern the dynamics of the radiation belts (radial, pitch angle and energy diffusion) but rather our limitations in specifying accurate boundary conditions and code inputs. We use here DREAM-3D runs to show the sensitivity of the modeled outcomes to these boundary conditions and inputs, and also discuss alternate "proxy" approaches to obtain the required inputs from other (ground-based) sources.

  3. Registration of 3D and multispectral data for the study of cultural heritage surfaces.

    PubMed

    Chane, Camille Simon; Schütze, Rainer; Boochs, Frank; Marzani, Franck S

    2013-01-01

    We present a technique for the multi-sensor registration of featureless datasets based on the photogrammetric tracking of the acquisition systems in use. This method is developed for the in situ study of cultural heritage objects and is tested by digitizing a small canvas successively with a 3D digitization system and a multispectral camera while simultaneously tracking the acquisition systems with four cameras and using a cubic target frame with a side length of 500 mm. The achieved tracking accuracy is better than 0.03 mm spatially and 0.150 mrad angularly. This allows us to seamlessly register the 3D acquisitions and to project the multispectral acquisitions on the 3D model. PMID:23322103

  4. 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation.

    PubMed

    Smeriglio, Piera; Lai, Janice H; Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development. PMID:26484414

  5. 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

    PubMed Central

    Yang, Fan; Bhutani, Nidhi

    2015-01-01

    Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development. PMID:26484414

  6. 3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

    SciTech Connect

    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, 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.

  7. New 3D-Culture Approaches to Study Interactions of Bone Marrow Adipocytes with Metastatic Prostate Cancer Cells

    PubMed Central

    Herroon, Mackenzie Katheryn; Diedrich, Jonathan Driscoll; Podgorski, Izabela

    2016-01-01

    Adipocytes are a major component of the bone marrow that can critically affect metastatic progression in bone. Understanding how the marrow fat cells influence growth, behavior, and survival of tumor cells requires utilization of in vitro cell systems that can closely mimic the physiological microenvironment. Herein, we present two new three-dimensional (3D) culture approaches to study adipocyte–tumor cell interactions in vitro. The first is a transwell-based system composed of the marrow-derived adipocytes in 3D collagen I gels and reconstituted basement membrane-overlayed prostate tumor cell spheroids. Tumor cells cultured under these 3D conditions are continuously exposed to adipocyte-derived factors, and their response can be evaluated by morphological and immunohistochemical analyses. We show via immunofluorescence analysis of metabolism-associated proteins that under 3D conditions tumor cells have significantly different metabolic response to adipocytes than tumor cells grown in 2D culture. We also demonstrate that this model allows for incorporation of other cell types, such as bone marrow macrophages, and utilization of dye-quenched collagen substrates for examination of proteolysis-driven responses to adipocyte- and macrophage-derived factors. Our second 3D culture system is designed to study tumor cell invasion toward the adipocytes and the consequent interaction between the two cell types. In this model, marrow adipocytes are separated from the fluorescently labeled tumor cells by a layer of collagen I. At designated time points, adipocytes are stained with BODIPY and confocal z-stacks are taken through the depth of the entire culture to determine the distance traveled between the two cell types over time. We demonstrate that this system can be utilized to study effects of candidate factors on tumor invasion toward the adipocytes. We also show that immunohistochemical analyses can be performed to evaluate the impact of direct interaction of prostate

  8. 3D Cultures of prostate cancer cells cultured in a novel high-throughput culture platform are more resistant to chemotherapeutics compared to cells cultured in monolayer.

    PubMed

    Chambers, Karen F; Mosaad, Eman M O; Russell, Pamela J; Clements, Judith A; Doran, Michael R

    2014-01-01

    Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing. PMID:25380249

  9. Preclinical validation and imaging of Wnt-induced repair in human 3D lung tissue cultures.

    PubMed

    Uhl, Franziska E; Vierkotten, Sarah; Wagner, Darcy E; Burgstaller, Gerald; Costa, Rita; Koch, Ina; Lindner, Michael; Meiners, Silke; Eickelberg, Oliver; Königshoff, Melanie

    2015-10-01

    Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/β-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3β inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/β-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/β-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/β-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/β-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs. PMID:25929950

  10. Bridging the Gap: From 2D Cell Culture to 3D Microengineered Extracellular Matrices.

    PubMed

    Li, Yanfen; Kilian, Kristopher A

    2015-12-30

    Historically the culture of mammalian cells in the laboratory has been performed on planar substrates with media cocktails that are optimized to maintain phenotype. However, it is becoming increasingly clear that much of biology discerned from 2D studies does not translate well to the 3D microenvironment. Over the last several decades, 2D and 3D microengineering approaches have been developed that better recapitulate the complex architecture and properties of in vivo tissue. Inspired by the infrastructure of the microelectronics industry, lithographic patterning approaches have taken center stage because of the ease in which cell-sized features can be engineered on surfaces and within a broad range of biocompatible materials. Patterning and templating techniques enable precise control over extracellular matrix properties including: composition, mechanics, geometry, cell-cell contact, and diffusion. In this review article we explore how the field of engineered extracellular matrices has evolved with the development of new hydrogel chemistry and the maturation of micro- and nano- fabrication. Guided by the spatiotemporal regulation of cell state in developing tissues, techniques for micropatterning in 2D, pseudo-3D systems, and patterning within 3D hydrogels will be discussed in the context of translating the information gained from 2D systems to synthetic engineered 3D tissues. PMID:26592366

  11. A 3D aligned microfibrous myocardial tissue construct cultured under transient perfusion.

    PubMed

    Kenar, Halime; Kose, Gamze T; Toner, Mehmet; Kaplan, David L; Hasirci, Vasif

    2011-08-01

    The goal of this study was to design and develop a myocardial patch to use in the repair of myocardial infarctions or to slow down tissue damage and improve long-term heart function. The basic 3D construct design involved two biodegradable macroporous tubes, to allow transport of growth media to the cells within the construct, and cell seeded, aligned fiber mats wrapped around them. The microfibrous mat housed mesenchymal stem cells (MSCs) from human umbilical cord matrix (Wharton's Jelly) aligned in parallel to each other in a similar way to cell organization in native myocardium. Aligned micron-sized fiber mats were obtained by electrospinning a polyester blend (PHBV (5% HV), P(L-D,L)LA (70:30) and poly(glycerol sebacate) (PGS)). The micron-sized electrospun parallel fibers were effective in Wharton's Jelly (WJ) MSCs alignment and the cells were able to retract the mat. The 3D construct was cultured in a microbioreactor by perfusing the growth media transiently through the macroporous tubing for two weeks and examined by fluorescence microscopy for cell distribution and preservation of alignment. The fluorescence images of thin sections of 3D constructs from static and perfused cultures confirmed enhanced cell viability, uniform cell distribution and alignment due to nutrient provision from inside the 3D structure. PMID:21570112

  12. Mackay campus of environmental education and digital cultural construction: the application of 3D virtual reality

    NASA Astrophysics Data System (ADS)

    Chien, Shao-Chi; Chung, Yu-Wei; Lin, Yi-Hsuan; Huang, Jun-Yi; Chang, Jhih-Ting; He, Cai-Ying; Cheng, Yi-Wen

    2012-04-01

    This study uses 3D virtual reality technology to create the "Mackay campus of the environmental education and digital cultural 3D navigation system" for local historical sites in the Tamsui (Hoba) area, in hopes of providing tourism information and navigation through historical sites using a 3D navigation system. We used Auto CAD, Sketch Up, and SpaceEyes 3D software to construct the virtual reality scenes and create the school's historical sites, such as the House of Reverends, the House of Maidens, the Residence of Mackay, and the Education Hall. We used this technology to complete the environmental education and digital cultural Mackay campus . The platform we established can indeed achieve the desired function of providing tourism information and historical site navigation. The interactive multimedia style and the presentation of the information will allow users to obtain a direct information response. In addition to showing the external appearances of buildings, the navigation platform can also allow users to enter the buildings to view lifelike scenes and textual information related to the historical sites. The historical sites are designed according to their actual size, which gives users a more realistic feel. In terms of the navigation route, the navigation system does not force users along a fixed route, but instead allows users to freely control the route they would like to take to view the historical sites on the platform.

  13. Genetically Encoded Sender–Receiver System in 3D Mammalian Cell Culture

    PubMed Central

    2013-01-01

    Engineering spatial patterning in mammalian cells, employing entirely genetically encoded components, requires solving several problems. These include how to code secreted activator or inhibitor molecules and how to send concentration-dependent signals to neighboring cells, to control gene expression. The Madin–Darby Canine Kidney (MDCK) cell line is a potential engineering scaffold as it forms hollow spheres (cysts) in 3D culture and tubulates in response to extracellular hepatocyte growth factor (HGF). We first aimed to graft a synthetic patterning system onto single developing MDCK cysts. We therefore developed a new localized transfection method to engineer distinct sender and receiver regions. A stable reporter line enabled reversible EGFP activation by HGF and modulation by a secreted repressor (a truncated HGF variant, NK4). By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression. Together, these components provide a toolkit for engineering cell–cell communication networks in 3D cell culture. PMID:24313393

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  15. Preparation of 3D fibrin scaffolds for stem cell culture applications.

    PubMed

    Kolehmainen, Kathleen; Willerth, Stephanie M

    2012-01-01

    Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the

  16. Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

    PubMed Central

    Kolehmainen, Kathleen; Willerth, Stephanie M.

    2012-01-01

    Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis

  17. MAPLE deposition of 3D micropatterned polymeric substrates for cell culture

    NASA Astrophysics Data System (ADS)

    Paun, Irina Alexandra; Mihailescu, Mona; Calenic, Bogdan; Luculescu, Catalin Romeo; Greabu, Maria; Dinescu, Maria

    2013-08-01

    3D micropatterned poly(lactide-co-glycolide)/polyurethane (PLGA/PU) substrates were produced by MAPLE deposition through masks and used for regulating the behavior of oral keratinocyte stem cells in response to topography. Flat PLGA/PU substrates were produced for comparison. 3D imaging of the PLGA/PU substrates and of the cultured cells was performed by Digital Holographic Microscopy. The micropatterns were in the shape of squares of 50 × 50 and 80 × 80 μm2 areas, ~1.8 μm in height and separated by 20 μm wide channels. It was found that substrate topography guided the adhesion of the cultured cells: on the smooth substrates the cells adhered randomly and showed no preferred orientation; in contrast, on the micropatterned substrates the cells adhered preferentially onto the squares and not in the separating channels. Furthermore, key properties of the cells (size, viability, proliferation rate and stem cell marker expression) did not show any dependence on substrate topography. The size of the cultured cells, their viability, the proportions of actively/slow proliferating cells, as well as the stem cell markers expressions, were similar for both flat and micropatterned substrates. Finally, it was found that the cells cultured on the PLGA/PU substrates deposited by MAPLE exhibited similar properties as the controls (i.e. cells cultured on glass slides), indicating the capability of the former to preserve the properties of the keratinocyte stem cells.

  18. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-07-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation <10%) cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells.

  19. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures.

    PubMed

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-01-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation <10%) cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells. PMID:26153550

  20. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

    PubMed Central

    Chen, Yu-Chih; Lou, Xia; Zhang, Zhixiong; Ingram, Patrick; Yoon, Euisik

    2015-01-01

    Photodynamic therapy (PDT), wherein light sensitive non-toxic agents are locally and selectively activated using light, has emerged as an appealing alternative to traditional cancer chemotherapy. Yet to date, PDT efficacy has been mostly characterized using 2D cultures. Compared to 2D cultures, 3D sphere culture generates unique spatial distributions of nutrients and oxygen for the cells that better mimics the in-vivo conditions. Using a novel polyHEMA (non-adherent polymer) fabrication process, we developed a microfluidic sphere formation platform that can (1) generate 1,024 uniform (size variation <10%) cancer spheres within a 2 cm by 2 cm core area, (2) culture spheres for more than 2 weeks, and (3) allow the retrieval of spheres. Using the presented platform, we have successfully characterized the different responses in 2D and 3D cell culture to PDT. Furthermore, we investigated the treatment resistance effect in cancer cells induced by tumor associated fibroblasts (CAF). Although the CAFs can enhance the resistance to traditional chemotherapy agents, no significant difference in PDT was observed. The preliminary results suggest that the PDT can be an attractive alternative cancer therapy, which is less affected by the therapeutic resistance induced by cancer associated cells. PMID:26153550

  1. Video lensfree microscopy of 2D and 3D culture of cells

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Coutard, J.-G.; Cioni, O.; Momey, F.; Navarro, F.; Menneteau, M.; Chalmond, B.; Obeid, P.; Haguet, V.; David-Watine, B.; Dubrulle, N.; Shorte, S.; van der Sanden, B.; Di Natale, C.; Hamard, L.; Wion, D.; Dolega, M. E.; Picollet-D'hahan, N.; Gidrol, X.; Dinten, J.-M.

    2014-03-01

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.

  2. Dynamic perfusion bioreactor system for 3D culture of rat bone marrow mesenchymal stem cells on nanohydroxyapatite/polyamide 66 scaffold in vitro.

    PubMed

    Qian, Xu; Yuan, Fang; Zhimin, Zhu; Anchun, Mo

    2013-08-01

    The aim of the study was to investigate the biocompatibility and osteogenic effectiveness of the porous nanohydroxyapatite/polyamide 66 (n-HA/PA66) scaffold material that was cultured with the rat bone marrow mesenchymal stem cells (rBMSCs), under the static culture condition and the dynamic perfusion culture condition in vitro, and to investigate whether the 3D perfusion culture condition was better in provoking proliferation of rBMSCs than the 3D static culture condition. The Methyl thiazolyl tetrazolium (MTT) assay, alkaline phosphatase (ALP) activity assay, Osteocalcin (OCN) assay and scanning electron microscope (SEM) were used to observe the proliferation and differentiation of rBMSCs. The samples were respectively harvested at 1st, 3rd, 7th, 14th, and 21st days and effect comparisons were made between the two of the culture conditions. The results showed that values of MTT, ALP, and OCN were increased continuously and revealed a significant difference between the two culture conditions (p < 0.05). On the 14th day, SEM revealed calcified nodules 2-8 μm in diameter in the lamellar structure. Under the static culture condition, the pores were covered with the cells looking like a piece of blanket, but under the perfusion culture condition the cells were observed to have a 3D lamellar structure. In conclusion, the porous n-HA/PA66 scaffold material can be used as a good candidate material for the bone scaffold construction in the tissue engineering because of its excellent 3D structure, which can greatly improve the proliferation and differentiation of rBMSCs and make them proliferate and osteogenesis even better under the perfusion culture condition. PMID:23362119

  3. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.

    PubMed

    Knight, Eleanor; Przyborski, Stefan

    2015-12-01

    Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science. PMID:25411113

  4. Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel.

    PubMed

    Farnsworth, Nikki L; Mead, Benjamin E; Antunez, Lorena R; Palmer, Amy E; Bryant, Stephanie J

    2014-11-01

    The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca(2+)]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca(2+) chelator BAPTA-AM decreased GAG synthesis by 32-46% and was similar among all conditions, suggesting multiple roles for Ca(2+) mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca(2+)]i signaling as a potential mediator. PMID:25128592

  5. Quantification of Dynamic Morphological Drug Responses in 3D Organotypic Cell Cultures by Automated Image Analysis

    PubMed Central

    Härmä, Ville; Schukov, Hannu-Pekka; Happonen, Antti; Ahonen, Ilmari; Virtanen, Johannes; Siitari, Harri; Åkerfelt, Malin; Lötjönen, Jyrki; Nees, Matthias

    2014-01-01

    Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports

  6. Minimal camera networks for 3D image based modeling of cultural heritage objects.

    PubMed

    Alsadik, Bashar; Gerke, Markus; Vosselman, George; Daham, Afrah; Jasim, Luma

    2014-01-01

    3D modeling of cultural heritage objects like artifacts, statues and buildings is nowadays an important tool for virtual museums, preservation and restoration. In this paper, we introduce a method to automatically design a minimal imaging network for the 3D modeling of cultural heritage objects. This becomes important for reducing the image capture time and processing when documenting large and complex sites. Moreover, such a minimal camera network design is desirable for imaging non-digitally documented artifacts in museums and other archeological sites to avoid disturbing the visitors for a long time and/or moving delicate precious objects to complete the documentation task. The developed method is tested on the Iraqi famous statue "Lamassu". Lamassu is a human-headed winged bull of over 4.25 m in height from the era of Ashurnasirpal II (883-859 BC). Close-range photogrammetry is used for the 3D modeling task where a dense ordered imaging network of 45 high resolution images were captured around Lamassu with an object sample distance of 1 mm. These images constitute a dense network and the aim of our study was to apply our method to reduce the number of images for the 3D modeling and at the same time preserve pre-defined point accuracy. Temporary control points were fixed evenly on the body of Lamassu and measured by using a total station for the external validation and scaling purpose. Two network filtering methods are implemented and three different software packages are used to investigate the efficiency of the image orientation and modeling of the statue in the filtered (reduced) image networks. Internal and external validation results prove that minimal image networks can provide highly accurate records and efficiency in terms of visualization, completeness, processing time (>60% reduction) and the final accuracy of 1 mm. PMID:24670718

  7. Minimal Camera Networks for 3D Image Based Modeling of Cultural Heritage Objects

    PubMed Central

    Alsadik, Bashar; Gerke, Markus; Vosselman, George; Daham, Afrah; Jasim, Luma

    2014-01-01

    3D modeling of cultural heritage objects like artifacts, statues and buildings is nowadays an important tool for virtual museums, preservation and restoration. In this paper, we introduce a method to automatically design a minimal imaging network for the 3D modeling of cultural heritage objects. This becomes important for reducing the image capture time and processing when documenting large and complex sites. Moreover, such a minimal camera network design is desirable for imaging non-digitally documented artifacts in museums and other archeological sites to avoid disturbing the visitors for a long time and/or moving delicate precious objects to complete the documentation task. The developed method is tested on the Iraqi famous statue “Lamassu”. Lamassu is a human-headed winged bull of over 4.25 m in height from the era of Ashurnasirpal II (883–859 BC). Close-range photogrammetry is used for the 3D modeling task where a dense ordered imaging network of 45 high resolution images were captured around Lamassu with an object sample distance of 1 mm. These images constitute a dense network and the aim of our study was to apply our method to reduce the number of images for the 3D modeling and at the same time preserve pre-defined point accuracy. Temporary control points were fixed evenly on the body of Lamassu and measured by using a total station for the external validation and scaling purpose. Two network filtering methods are implemented and three different software packages are used to investigate the efficiency of the image orientation and modeling of the statue in the filtered (reduced) image networks. Internal and external validation results prove that minimal image networks can provide highly accurate records and efficiency in terms of visualization, completeness, processing time (>60% reduction) and the final accuracy of 1 mm. PMID:24670718

  8. Towards a 3d Based Platform for Cultural Heritage Site Survey and Virtual Exploration

    NASA Astrophysics Data System (ADS)

    Seinturier, J.; Riedinger, C.; Mahiddine, A.; Peloso, D.; Boï, J.-M.; Merad, D.; Drap, P.

    2013-07-01

    This paper present a 3D platform that enables to make both cultural heritage site survey and its virtual exploration. It provides a single and easy way to use framework for merging multi scaled 3D measurements based on photogrammetry, documentation produced by experts and the knowledge of involved domains leaving the experts able to extract and choose the relevant information to produce the final survey. Taking into account the interpretation of the real world during the process of archaeological surveys is in fact the main goal of a survey. New advances in photogrammetry and the capability to produce dense 3D point clouds do not solve the problem of surveys. New opportunities for 3D representation are now available and we must to use them and find new ways to link geometry and knowledge. The new platform is able to efficiently manage and process large 3D data (points set, meshes) thanks to the implementation of space partition methods coming from the state of the art such as octrees and kd-trees and thus can interact with dense point clouds (thousands to millions of points) in real time. The semantisation of raw 3D data relies on geometric algorithms such as geodetic path computation, surface extraction from dense points cloud and geometrical primitive optimization. The platform provide an interface that enables expert to describe geometric representations of interesting objects like ashlar blocs, stratigraphic units or generic items (contour, lines, … ) directly onto the 3D representation of the site and without explicit links to underlying algorithms. The platform provide two ways for describing geometric representation. If oriented photographs are available, the expert can draw geometry on a photograph and the system computes its 3D representation by projection on the underlying mesh or the points cloud. If photographs are not available or if the expert wants to only use the 3D representation then he can simply draw objects shape on it. When 3D

  9. Optimization of a 3D Dynamic Culturing System for In Vitro Modeling of Frontotemporal Neurodegeneration-Relevant Pathologic Features

    PubMed Central

    Tunesi, Marta; Fusco, Federica; Fiordaliso, Fabio; Corbelli, Alessandro; Biella, Gloria; Raimondi, Manuela T.

    2016-01-01

    Frontotemporal lobar degeneration (FTLD) is a severe neurodegenerative disorder that is diagnosed with increasing frequency in clinical setting. Currently, no therapy is available and in addition the molecular basis of the disease are far from being elucidated. Consequently, it is of pivotal importance to develop reliable and cost-effective in vitro models for basic research purposes and drug screening. To this respect, recent results in the field of Alzheimer’s disease have suggested that a tridimensional (3D) environment is an added value to better model key pathologic features of the disease. Here, we have tried to add complexity to the 3D cell culturing concept by using a microfluidic bioreactor, where cells are cultured under a continuous flow of medium, thus mimicking the interstitial fluid movement that actually perfuses the body tissues, including the brain. We have implemented this model using a neuronal-like cell line (SH-SY5Y), a widely exploited cell model for neurodegenerative disorders that shows some basic features relevant for FTLD modeling, such as the release of the FTLD-related protein progranulin (PRGN) in specific vesicles (exosomes). We have efficiently seeded the cells on 3D scaffolds, optimized a disease-relevant oxidative stress experiment (by targeting mitochondrial function that is one of the possible FTLD-involved pathological mechanisms) and evaluated cell metabolic activity in dynamic culture in comparison to static conditions, finding that SH-SY5Y cells cultured in 3D scaffold are susceptible to the oxidative damage triggered by a mitochondrial-targeting toxin (6-OHDA) and that the same cells cultured in dynamic conditions kept their basic capacity to secrete PRGN in exosomes once recovered from the bioreactor and plated in standard 2D conditions. We think that a further improvement of our microfluidic system may help in providing a full device where assessing basic FTLD-related features (including PRGN dynamic secretion) that may

  10. 3D culture of ovarian follicles: a system towards their engineering?

    PubMed

    Zuccotti, Maurizio; Merico, Valeria; Rebuzzini, Paola; Belli, Martina; Vigone, Giulia; Mulas, Francesca; Fassina, Lorenzo; Wruck, Wasco; Adjaye, James; Bellazzi, Riccardo; Garagna, Silvia

    2015-01-01

    Infertility in women is a health priority. Designing a robust culture protocol capable of attaining complete follicle growth is an exciting challenge, for its potential clinical applications, but also as a model to observe and closely study the sequence of molecular events that lie behind the intricate relationship existing between the oocyte and surrounding follicle cells. Here, we describe the procedures used to maintain the ovarian follicle 3D architecture employing a variety of in vitro systems and several types of matrices. Collagen and alginate are the matrices that led to better results, including proof-of-concept of full-term development. Pioneer in its kind, these studies underlie the drawbacks encountered and the need for a culture system that allows more quantitative analyses and predictions, projecting the culture of the ovarian follicle into the realm of tissue engineering. PMID:26505254

  11. Optimal 3-D culture of primary articular chondrocytes for use in the Rotating Wall Vessel Bioreactor

    PubMed Central

    Mellor, Liliana F.; Baker, Travis L.; Brown, Raquel J.; Catlin, Lindsey W.; Oxford, Julia Thom

    2014-01-01

    INTRODUCTION Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology but also maintain gene expression characteristics of primary articular chondrocytes. METHODS Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. RESULTS Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 days. DISCUSSION Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering. PMID:25199120

  12. Heat Transfer Boundary Conditions in the RELAP5-3D Code

    SciTech Connect

    Richard A. Riemke; Cliff B. Davis; Richard R. Schultz

    2008-05-01

    The heat transfer boundary conditions used in the RELAP5-3D computer program have evolved over the years. Currently, RELAP5-3D has the following options for the heat transfer boundary conditions: (a) heat transfer correlation package option, (b) non-convective option (from radiation/conduction enclosure model or symmetry/insulated conditions), and (c) other options (setting the surface temperature to a volume fraction averaged fluid temperature of the boundary volume, obtaining the surface temperature from a control variable, obtaining the surface temperature from a time-dependent general table, obtaining the heat flux from a time-dependent general table, or obtaining heat transfer coefficients from either a time- or temperature-dependent general table). These options will be discussed, including the more recent ones.

  13. Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle

    PubMed Central

    Zaman, Nishat; Cole, Darren J.; Walker, Matthew J.; Legant, Wesley R.; Boudou, Thomas; Chen, Christopher S.; Favreau, John T.; Gaudette, Glenn R.; Cowley, Elizabeth A.; Maksym, Geoffrey N.

    2013-01-01

    Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell “microtissues” capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma. PMID:23125251

  14. Biodynamic Doppler imaging of subcellular motion inside 3D living tissue culture and biopsies (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nolte, David D.

    2016-03-01

    Biodynamic imaging is an emerging 3D optical imaging technology that probes up to 1 mm deep inside three-dimensional living tissue using short-coherence dynamic light scattering to measure the intracellular motions of cells inside their natural microenvironments. Biodynamic imaging is label-free and non-invasive. The information content of biodynamic imaging is captured through tissue dynamics spectroscopy that displays the changes in the Doppler signatures from intracellular constituents in response to applied compounds. The affected dynamic intracellular mechanisms include organelle transport, membrane undulations, cytoskeletal restructuring, strain at cellular adhesions, cytokinesis, mitosis, exo- and endo-cytosis among others. The development of 3D high-content assays such as biodynamic profiling can become a critical new tool for assessing efficacy of drugs and the suitability of specific types of tissue growth for drug discovery and development. The use of biodynamic profiling to predict clinical outcome of living biopsies to cancer therapeutics can be developed into a phenotypic companion diagnostic, as well as a new tool for therapy selection in personalized medicine. This invited talk will present an overview of the optical, physical and physiological processes involved in biodynamic imaging. Several different biodynamic imaging modalities include motility contrast imaging (MCI), tissue-dynamics spectroscopy (TDS) and tissue-dynamics imaging (TDI). A wide range of potential applications will be described that include process monitoring for 3D tissue culture, drug discovery and development, cancer therapy selection, embryo assessment for in-vitro fertilization and artificial reproductive technologies, among others.

  15. 3D cell culture systems modeling tumor growth determinants in cancer target discovery.

    PubMed

    Thoma, Claudio R; Zimmermann, Miriam; Agarkova, Irina; Kelm, Jens M; Krek, Wilhelm

    2014-04-01

    Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models. PMID:24636868

  16. A versatile 3D culture model facilitates monitoring of astrocytes undergoing reactive gliosis

    PubMed Central

    East, Emma; Golding, Jonathan P; Phillips, James B

    2009-01-01

    A major impediment to CNS repair is the glial scar, which forms following damage and is composed mainly of ramified, ‘reactive’ astrocytes that inhibit neuronal regrowth. The transition of astrocytes into this reactive phenotype (reactive gliosis) is a potential therapeutic target, but glial scar formation has proved difficult to study in monolayer cultures because they induce constitutive astrocyte activation. Here we demonstrate a 3D collagen gel system in which primary rat astrocytes were maintained in a persistently less reactive state than comparable cells in monolayer, resembling their status in the undamaged CNS. Reactivity, proliferation and viability were monitored and quantified using confocal, fluorescence and time-lapse microscopy, 3D image analysis, RT–PCR and ELISA. To assess the potential of this system as a model of reactive gliosis, astrocytes in 3D were activated with TGFβ1 to a ramified, reactive phenotype (elevated GFAP, Aquaporin 4, CSPG, Vimentin and IL-6 secretion). This provides a versatile system in which astrocytes can be maintained in a resting state, then be triggered to undergo reactive gliosis, enabling real-time monitoring and quantitative analysis throughout and providing a powerful new tool for research into CNS damage and repair. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19813215

  17. Acrylic-acid-functionalized PolyHIPE scaffolds for use in 3D cell culture.

    PubMed

    Hayward, Adam S; Sano, Naoko; Przyborski, Stefan A; Cameron, Neil R

    2013-12-01

    This study describes the development of a functional porous polymer for use as a scaffold to support 3D hepatocyte culture. A high internal phase emulsion (HIPE) is prepared containing the monomers styrene (STY), divinylbenzene (DVB), and 2-ethylhexyl acrylate (EHA) in the external oil phase and the monomer acrylic acid (Aa) in the internal aqueous phase. Upon thermal polymerization with azobisisobutyronitrile (AIBN), the resulting porous polymer (polyHIPE) is found to have an open-cell morphology and a porosity of 89%, both suitable characteristics for 3D cell scaffold applications. X-ray photo-electron spectroscopy reveals that the polyHIPE surface contained 7.5% carboxylic acid functionality, providing a useful substrate for subsequent surface modifications and bio-conjugations. Initial bio-compatibility assessments with human hepatocytes show that the acid functionality does not have any detrimental effect on cell adhesion. It is therefore believed that this material can be a useful precursor scaffold towards 3D substrates that offer tailored surface functionality for enhanced cell adhesion. PMID:24243821

  18. Micro 3D cell culture systems for cellular behavior studies: Culture matrices, devices, substrates, and in-situ sensing methods.

    PubMed

    Choi, Jonghoon; Lee, Eun Kyu; Choo, Jaebum; Yuh, Junhan; Hong, Jong Wook

    2015-09-01

    Microfabricated systems equipped with 3D cell culture devices and in-situ cellular biosensing tools can be a powerful bionanotechnology platform to investigate a variety of biomedical applications. Various construction substrates such as plastics, glass, and paper are used for microstructures. When selecting a construction substrate, a key consideration is a porous microenvironment that allows for spheroid growth and mimics the extracellular matrix (ECM) of cell aggregates. Various bio-functionalized hydrogels are ideal candidates that mimic the natural ECM for 3D cell culture. When selecting an optimal and appropriate microfabrication method, both the intended use of the system and the characteristics and restrictions of the target cells should be carefully considered. For highly sensitive and near-cell surface detection of excreted cellular compounds, SERS-based microsystems capable of dual modal imaging have the potential to be powerful tools; however, the development of optical reporters and nanoprobes remains a key challenge. We expect that the microsystems capable of both 3D cell culture and cellular response monitoring would serve as excellent tools to provide fundamental cellular behavior information for various biomedical applications such as metastasis, wound healing, high throughput screening, tissue engineering, regenerative medicine, and drug discovery and development. PMID:26358782

  19. Advanced quadratures and periodic boundary conditions in parallel 3D S{sub n} transport

    SciTech Connect

    Manalo, K.; Yi, C.; Huang, M.; Sjoden, G.

    2013-07-01

    Significant updates in numerical quadratures have warranted investigation with 3D Sn discrete ordinates transport. We show new applications of quadrature departing from level symmetric (S{sub 2}o). investigating 3 recently developed quadratures: Even-Odd (EO), Linear-Discontinuous Finite Element - Surface Area (LDFE-SA), and the non-symmetric Icosahedral Quadrature (IC). We discuss implementation changes to 3D Sn codes (applied to Hybrid MOC-Sn TITAN and 3D parallel PENTRAN) that can be performed to accommodate Icosahedral Quadrature, as this quadrature is not 90-degree rotation invariant. In particular, as demonstrated using PENTRAN, the properties of Icosahedral Quadrature are suitable for trivial application using periodic BCs versus that of reflective BCs. In addition to implementing periodic BCs for 3D Sn PENTRAN, we implemented a technique termed 'angular re-sweep' which properly conditions periodic BCs for outer eigenvalue iterative loop convergence. As demonstrated by two simple transport problems (3-group fixed source and 3-group reflected/periodic eigenvalue pin cell), we remark that all of the quadratures we investigated are generally superior to level symmetric quadrature, with Icosahedral Quadrature performing the most efficiently for problems tested. (authors)

  20. Self-organization of neural patterns and structures in 3D culture of stem cells

    NASA Astrophysics Data System (ADS)

    Sasai, Yoshiki

    2013-05-01

    Over the last several years, much progress has been made for in vitro culture of mouse and human ES cells. Our laboratory focuses on the molecular and cellular mechanisms of neural differentiation from pluripotent cells. Pluripotent cells first become committed to the ectodermal fate and subsequently differentiate into uncommitted neuroectodermal cells. Both previous mammalian and amphibian studies on pluripotent cells have indicated that the neural fate is a sort of the basal direction of the differentiation of these cells while mesoendodermal differentiation requires extrinsic inductive signals. ES cells differentiate into neuroectodermal cells with a rostral-most character (telencephalon and hypothalamus) when they are cultured in the absence of strong patterning signals. In this talk, I first discuss this issue by referring to our recent data on the mechanism of spontaneous neural differentiation in serum-free culture of mouse ES cells. Then, I will talk about self-organization phenomena observed in 3D culture of ES cells, which lead to tissue-autonomous formation of regional structures such as layered cortical tissues. I also discuss our new attempt to monitor these in vitro morphogenetic processes by live imaging, in particular, self-organizing morphogenesis of the optic cup in three-dimensional cultures.

  1. Inflow/Outflow Boundary Conditions with Application to FUN3D

    NASA Technical Reports Server (NTRS)

    Carlson, Jan-Renee

    2011-01-01

    Several boundary conditions that allow subsonic and supersonic flow into and out of the computational domain are discussed. These boundary conditions are demonstrated in the FUN3D computational fluid dynamics (CFD) code which solves the three-dimensional Navier-Stokes equations on unstructured computational meshes. The boundary conditions are enforced through determination of the flux contribution at the boundary to the solution residual. The boundary conditions are implemented in an implicit form where the Jacobian contribution of the boundary condition is included and is exact. All of the flows are governed by the calorically perfect gas thermodynamic equations. Three problems are used to assess these boundary conditions. Solution residual convergence to machine zero precision occurred for all cases. The converged solution boundary state is compared with the requested boundary state for several levels of mesh densities. The boundary values converged to the requested boundary condition with approximately second-order accuracy for all of the cases.

  2. Individual versus Collective Fibroblast Spreading and Migration: Regulation by Matrix Composition in 3-D Culture

    PubMed Central

    Miron-Mendoza, Miguel; Lin, Xihui; Ma, Lisha; Ririe, Peter; Petroll, W. Matthew

    2012-01-01

    Extracellular matrix (ECM) supplies both physical and chemical signals to cells and provides a substrate through which fibroblasts migrate during wound repair. To directly assess how ECM composition regulates this process, we used a nested 3D matrix model in which cell-populated collagen buttons were embedded in cell-free collagen or fibrin matrices. Time-lapse microscopy was used to record the dynamic pattern of cell migration into the outer matrices, and 3-D confocal imaging was used to assess cell connectivity and cytoskeletal organization. Corneal fibroblasts stimulated with PDGF migrated more rapidly into collagen as compared to fibrin. In addition, the pattern of fibroblast migration into fibrin and collagen ECMs was strikingly different. Corneal fibroblasts migrating into collagen matrices developed dendritic processes and moved independently, whereas cells migrating into fibrin matrices had a more fusiform morphology and formed an interconnected meshwork. A similar pattern was observed when using dermal fibroblasts, suggesting that this response in not unique to corneal cells. We next cultured corneal fibroblasts within and on top of standard collagen and fibrin matrices to assess the impact of ECM composition on the cell spreading response. Similar differences in cell morphology and connectivity were observed – cells remained separated on collagen but coalesced into clusters on fibrin. Cadherin was localized to junctions between interconnected cells, whereas fibronectin was present both between cells and at the tips of extending cell processes. Cells on fibrin matrices also developed more prominent stress fibers than those on collagen matrices. Importantly, these spreading and migration patterns were consistently observed on both rigid and compliant substrates, thus differences in ECM mechanical stiffness were not the underlying cause. Overall, these results demonstrate for the first time that ECM protein composition alone (collagen vs. fibrin) can

  3. Efficient Use of Video for 3d Modelling of Cultural Heritage Objects

    NASA Astrophysics Data System (ADS)

    Alsadik, B.; Gerke, M.; Vosselman, G.

    2015-03-01

    Currently, there is a rapid development in the techniques of the automated image based modelling (IBM), especially in advanced structure-from-motion (SFM) and dense image matching methods, and camera technology. One possibility is to use video imaging to create 3D reality based models of cultural heritage architectures and monuments. Practically, video imaging is much easier to apply when compared to still image shooting in IBM techniques because the latter needs a thorough planning and proficiency. However, one is faced with mainly three problems when video image sequences are used for highly detailed modelling and dimensional survey of cultural heritage objects. These problems are: the low resolution of video images, the need to process a large number of short baseline video images and blur effects due to camera shake on a significant number of images. In this research, the feasibility of using video images for efficient 3D modelling is investigated. A method is developed to find the minimal significant number of video images in terms of object coverage and blur effect. This reduction in video images is convenient to decrease the processing time and to create a reliable textured 3D model compared with models produced by still imaging. Two experiments for modelling a building and a monument are tested using a video image resolution of 1920×1080 pixels. Internal and external validations of the produced models are applied to find out the final predicted accuracy and the model level of details. Related to the object complexity and video imaging resolution, the tests show an achievable average accuracy between 1 - 5 cm when using video imaging, which is suitable for visualization, virtual museums and low detailed documentation.

  4. Low-level laser therapy in 3D cell culture model using gingival fibroblasts.

    PubMed

    Basso, Fernanda G; Soares, Diana G; de Souza Costa, Carlos Alberto; Hebling, Josimeri

    2016-07-01

    Besides extensive data about the effects of low-level laser therapy (LLLT) on different cell types, so far, these results were obtained from monolayer cell culture models, which have limitations in terms of cell morphology and phenotype expression. Therefore, for better in vitro evaluation of the effects of LLLT, this study was performed with a 3D cell culture model, where gingival fibroblasts were seeded in collagen matrix. Cells isolated from a healthy patient were seeded in wells of 24-well plates with culture medium (DMEM) supplemented with 10 % fetal bovine serum and collagen type I solution. After 5 days, a serum-free DMEM was added to the matrices with cells that were subjected or not to three consecutive irradiations of LLLT by means of the LaserTABLE diode device (780 nm, 25 mW) at 0.5, 1.5, and 3 J/cm(2). Twenty-four hours after the last irradiation, cell viability and morphology as well as gene expression of growth factors were assessed. Histological evaluation of matrices demonstrated uniform distribution and morphology of gingival fibroblasts within the collagen matrix. LLLT at 3 J/cm(2) increased gingival fibroblast viability. Enhanced gene expression of hCOL-I and hEGF was observed for 0.5 J/cm(2), while no significant changes were detected for the other irradiation densities tested. In conclusion, LLLT promoted biostimulation of gingival fibroblasts seeded in a 3D cell culture model, demonstrating that this model can be applied for phototherapy studies and that LLLT could penetrate the collagen matrix to increase cell functions related to tissue repair. PMID:27126408

  5. The Niha Sites (lebanon) Cultural Landscape: a 3d Model of Sanctuaries and Their Context

    NASA Astrophysics Data System (ADS)

    Yasmine, J.

    2013-07-01

    The paper aims at presenting the historical sites of Niha (Beqaa valley, Lebanon), their cultural values, and the methodology applied in the assessment of these values through the use of 3D modelling. The whole cultural landscape comprises the current village of Niha (altitude 1100 m), the archaeological site of Hosn-Niha (altitude 1350m), and the area located between these two sites. Two rural sanctuaries constitute the major archaeological remains present in the landscape: the first, located in the village of Niha, is composed of two roman temples with various archaeological structures; the second is located at the top of an antique settlement 2,5 km above the village of Niha. This second sanctuary Hosn-Niha, includes two temples, one church, remnants of numerous structures, and remains of an antique village. The cultural and religious values of both these sites are clear. However, questions arise regarding the choice for establishing the sanctuaries in these locations. The aim of the research is to try to understand the reasons for the various settlements in relationship with the topography and the landscape. The methodology applied in the research addresses two levels: a - The landscape level, and b - the built-up archaeology level. The global 3D models of both the landscape and the sanctuaries allow us to understand the various relations between the landscape, the sanctuaries and the various archaeological structures. An assessment of the various cultural resources found around the sanctuaries, while considering the reasons for their specific placement in the landscape can shed light on the reasons of these choices.

  6. Bioactive fish collagen/polycaprolactone composite nanofibrous scaffolds fabricated by electrospinning for 3D cell culture.

    PubMed

    Choi, Da Jeong; Choi, Seung Mi; Kang, Hae Yeong; Min, Hye-Jin; Lee, Rira; Ikram, Muhammad; Subhan, Fazli; Jin, Song Wan; Jeong, Young Hun; Kwak, Jong-Young; Yoon, Sik

    2015-07-10

    One of the most challenging objectives of 3D cell culture is the development of scaffolding materials with outstanding biocompatibility and favorable mechanical strength. In this study, we fabricated a novel nanofibrous scaffold composed of fish collagen (FC) and polycaprolactone (PCL) blends by using the electrospinning method. Nanofibrous scaffolds were characterized using a scanning electron microscope (SEM), and it was revealed that the diameter of nanofibers decreased as FC content was increased in the FC/PCL composite nanofibers. The cytocompatibility of the FC/PCL scaffolds was evaluated by SEM, WST-1 assay, confocal microscopy, western blot, and RT-PCR. It was found that the scaffolds not only facilitated the adhesion, spreading, protrusions, and proliferation of thymic epithelial cells (TECs), but also stimulated the expression of genes and proteins involved in cell adhesion and T-cell development. Thus, these results suggest that the FC/PCL composite nanofibrous scaffolds will be a useful model of 3D cell culture for TECs and may have wide applicability in the future for engineering tissues or organs. PMID:25617682

  7. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

    PubMed Central

    Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G.

    2016-01-01

    This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

  8. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures.

    PubMed

    Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G

    2016-01-01

    This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

  9. Quantitative Assessment of Local Collagen Matrix Remodeling in 3-D Culture: The Role of Rho Kinase

    PubMed Central

    Kim, Areum; Lakshman, Neema; Petroll, W.Matthew

    2007-01-01

    The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100 μm thick fibrillar collagen matrices and cultured for 24 hours in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p < 0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction. PMID:16978606

  10. a Semi-Automated Point Cloud Processing Methodology for 3d Cultural Heritage Documentation

    NASA Astrophysics Data System (ADS)

    Kıvılcım, C. Ö.; Duran, Z.

    2016-06-01

    The preliminary phase in any architectural heritage project is to obtain metric measurements and documentation of the building and its individual elements. On the other hand, conventional measurement techniques require tremendous resources and lengthy project completion times for architectural surveys and 3D model production. Over the past two decades, the widespread use of laser scanning and digital photogrammetry have significantly altered the heritage documentation process. Furthermore, advances in these technologies have enabled robust data collection and reduced user workload for generating various levels of products, from single buildings to expansive cityscapes. More recently, the use of procedural modelling methods and BIM relevant applications for historic building documentation purposes has become an active area of research, however fully automated systems in cultural heritage documentation still remains open. In this paper, we present a semi-automated methodology, for 3D façade modelling of cultural heritage assets based on parametric and procedural modelling techniques and using airborne and terrestrial laser scanning data. We present the contribution of our methodology, which we implemented in an open source software environment using the example project of a 16th century early classical era Ottoman structure, Sinan the Architect's Şehzade Mosque in Istanbul, Turkey.

  11. A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice

    PubMed Central

    Higuchi, Carolyn M.; Maeda, Yuuki; Horiuchi, Toshitaka; Yamazaki, Yukiko

    2015-01-01

    In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture

  12. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells.

    PubMed

    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O; Wagner, Mary B; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  13. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells

    PubMed Central

    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K.; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O.; Wagner, Mary B.; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  14. Defining an optimal surface chemistry for pluripotent stem cell culture in 2D and 3D

    NASA Astrophysics Data System (ADS)

    Zonca, Michael R., Jr.

    Surface chemistry is critical for growing pluripotent stem cells in an undifferentiated state. There is great potential to engineer the surface chemistry at the nanoscale level to regulate stem cell adhesion. However, the challenge is to identify the optimal surface chemistry of the substrata for ES cell attachment and maintenance. Using a high-throughput polymerization and screening platform, a chemically defined, synthetic polymer grafted coating that supports strong attachment and high expansion capacity of pluripotent stem cells has been discovered using mouse embryonic stem (ES) cells as a model system. This optimal substrate, N-[3-(Dimethylamino)propyl] methacrylamide (DMAPMA) that is grafted on 2D synthetic poly(ether sulfone) (PES) membrane, sustains the self-renewal of ES cells (up to 7 passages). DMAPMA supports cell attachment of ES cells through integrin beta1 in a RGD-independent manner and is similar to another recently reported polymer surface. Next, DMAPMA has been able to be transferred to 3D by grafting to synthetic, polymeric, PES fibrous matrices through both photo-induced and plasma-induced polymerization. These 3D modified fibers exhibited higher cell proliferation and greater expression of pluripotency markers of mouse ES cells than 2D PES membranes. Our results indicated that desirable surfaces in 2D can be scaled to 3D and that both surface chemistry and structural dimension strongly influence the growth and differentiation of pluripotent stem cells. Lastly, the feasibility of incorporating DMAPMA into a widely used natural polymer, alginate, has been tested. Novel adhesive alginate hydrogels have been successfully synthesized by either direct polymerization of DMAPMA and methacrylic acid blended with alginate, or photo-induced DMAPMA polymerization on alginate nanofibrous hydrogels. In particular, DMAPMA-coated alginate hydrogels support strong ES cell attachment, exhibiting a concentration dependency of DMAPMA. This research provides a

  15. Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

    PubMed Central

    Kim, Areum; Petroll, W. Matthew

    2007-01-01

    The purpose of this study was to investigate the role of microtubules in regulating corneal fibroblast structure and mechanical behavior using static (3-D) and dynamic (4-D) imaging of both cells and their surrounding matrix. Human corneal fibroblasts transfected to express GFP-zyxin (to label focal adhesions) or GFP-tubulin (to label microtubules) were plated at low density inside 100 μm thick type I collagen matrices. After 24 hours, the effects of nocodazole (to depolymerize microtubules), cytochalasin D (to disrupt f-actin), and/or Y-27632 (to block Rho-kinase) were evaluated using 3-D and 4-D imaging of both cells and ECM. After 24 hours of incubation, cells had well organized microtubules and prominent focal adhesions, and significant cell-induced matrix compaction was observed. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and additional cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process, when present. Following 24 hour exposure to nocodazole, there was an even greater increase in both the number of stress fibers and the amount of matrix compaction and alignment at the ends of cells. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture. PMID:17716657

  16. Dynamic Assessment of Fibroblast Mechanical Activity during Rac-induced Cell Spreading in 3-D Culture

    PubMed Central

    Petroll, W. Matthew; Ma, Lisha; Kim, Areum; Ly, Linda; Vishwanath, Mridula

    2009-01-01

    The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 μm thick fibrillar collagen matrices and cultured for 1 to 2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent. PMID:18452153

  17. Photopatterning of hydrogel scaffolds coupled to filter materials using stereolithography for perfused 3D culture of hepatocytes.

    PubMed

    Neiman, Jaclyn A Shepard; Raman, Ritu; Chan, Vincent; Rhoads, Mary G; Raredon, Micha Sam B; Velazquez, Jeremy J; Dyer, Rachel L; Bashir, Rashid; Hammond, Paula T; Griffith, Linda G

    2015-04-01

    In vitro models that recapitulate the liver's structural and functional complexity could prolong hepatocellular viability and function to improve platforms for drug toxicity studies and understanding liver pathophysiology. Here, stereolithography (SLA) was employed to fabricate hydrogel scaffolds with open channels designed for post-seeding and perfused culture of primary hepatocytes that form 3D structures in a bioreactor. Photopolymerizable polyethylene glycol-based hydrogels were fabricated coupled to chemically activated, commercially available filters (polycarbonate and polyvinylidene fluoride) using a chemistry that permitted cell viability, and was robust enough to withstand perfused culture of up to 1 µL/s for at least 7 days. SLA energy dose, photoinitiator concentrations, and pretreatment conditions were screened to determine conditions that maximized cell viability and hydrogel bonding to the filter. Multiple open channel geometries were readily achieved, and included ellipses and rectangles. Rectangular open channels employed for subsequent studies had final dimensions on the order of 350 µm by 850 µm. Cell seeding densities and flow rates that promoted cell viability were determined. Perfused culture of primary hepatocytes in hydrogel scaffolds in the presence of soluble epidermal growth factor (EGF) prolonged the maintenance of albumin production throughout the 7-day culture relative to 2D controls. This technique of bonding hydrogel scaffolds can be employed to fabricate soft scaffolds for a number of bioreactor configurations and applications. PMID:25384798

  18. A biofidelic 3D culture model to study the development of brain cellular systems.

    PubMed

    Ren, M; Du, C; Herrero Acero, E; Tang-Schomer, M D; Özkucur, N

    2016-01-01

    Little is known about how cells assemble as systems during corticogenesis to generate collective functions. We built a neurobiology platform that consists of fetal rat cerebral cortical cells grown within 3D silk scaffolds (SF). Ivermectin (Ivm), a glycine receptor (GLR) agonist, was used to modulate cell resting membrane potential (Vmem) according to methods described in a previous work that implicated Ivm in the arrangement and connectivity of cortical cell assemblies. The cells developed into distinct populations of neuroglial stem/progenitor cells, mature neurons or epithelial-mesenchymal cells. Importantly, the synchronized electrical activity in the newly developed cortical assemblies could be recorded as local field potential (LFP) measurements. This study therefore describes the first example of the development of a biologically relevant cortical plate assembly outside of the body. This model provides i) a preclinical basis for engineering cerebral cortex tissue autografts and ii) a biofidelic 3D culture model for investigating biologically relevant processes during the functional development of cerebral cortical cellular systems. PMID:27112667

  19. Primary human osteoblast culture on 3D porous collagen-hydroxyapatite scaffolds.

    PubMed

    Jones, Gemma L; Walton, Robin; Czernuszka, Jan; Griffiths, Sarah L; El Haj, Alicia J; Cartmell, Sarah H

    2010-09-15

    There is a need in tissue-engineering for 3D scaffolds that mimic the natural extracellular matrix of bone to enhance cell adhesion, proliferation, and differentiation. The scaffold is also required to be degradable. A highly porous scaffold has been developed to incorporate two of the extracellular components found in bone-collagen and hydroxyapatite (HA). The scaffold's collagen component is an afibrillar monomeric type I atelocollagen extracted from foetal calf's skin. This provided a novel environment for the inclusion of HA powder. Five hundred thousand primary human osteoblasts were seeded onto 4 mm cubed scaffolds that varied in ratio of HA to collagen. Weight ratios of 1:99, 25:75, 50:50, and 75:25 hydroxyapatite:collagen (HA:Collagen) were analysed. The scaffolds plus cells were cultured for 21 days. DNA assays and live/dead viability staining demonstrated that all of the scaffolds supported cell proliferation and viability. An alkaline phosphatase assay showed similar osteoblast phenotype maintenance on all of the 3D scaffolds analysed at 21 days. MicroCT analysis demonstrated an increase in total sample volume (correlating to increase in unmineralised matrix production). An even distribution of HA throughout the collagen matrix was observed using this technique. Also at 3 weeks, reductions in the percentage of the mineralised phase of the constructs were seen. These results indicate that each of the ratios of HA/collagen scaffolds have great potential for bone tissue engineering. PMID:20694991

  20. A biofidelic 3D culture model to study the development of brain cellular systems

    PubMed Central

    Ren, M.; Du, C.; Herrero Acero, E.; Tang-Schomer, M. D.; Özkucur, N.

    2016-01-01

    Little is known about how cells assemble as systems during corticogenesis to generate collective functions. We built a neurobiology platform that consists of fetal rat cerebral cortical cells grown within 3D silk scaffolds (SF). Ivermectin (Ivm), a glycine receptor (GLR) agonist, was used to modulate cell resting membrane potential (Vmem) according to methods described in a previous work that implicated Ivm in the arrangement and connectivity of cortical cell assemblies. The cells developed into distinct populations of neuroglial stem/progenitor cells, mature neurons or epithelial-mesenchymal cells. Importantly, the synchronized electrical activity in the newly developed cortical assemblies could be recorded as local field potential (LFP) measurements. This study therefore describes the first example of the development of a biologically relevant cortical plate assembly outside of the body. This model provides i) a preclinical basis for engineering cerebral cortex tissue autografts and ii) a biofidelic 3D culture model for investigating biologically relevant processes during the functional development of cerebral cortical cellular systems. PMID:27112667

  1. Interactions among Lung Cancer Cells, Fibroblasts, and Macrophages in 3D Co-Cultures and the Impact on MMP-1 and VEGF Expression

    PubMed Central

    Liu, Xiao-qing; Kiefl, Rosemarie; Roskopf, Claudia; Tian, Fei; Huber, Rudolf M.

    2016-01-01

    In vitro cell-based models of lung cancer are frequently employed to study invasion and the mechanisms behind metastasis. However, these models often study only one cell type with two-dimensional (2D) monolayer cell cultures, which do not accurately reflect the complexity of inflammation in vivo. Here, a three-dimensional (3D) cell co-culture collagen gel model was employed, containing human lung adenocarcinoma cells (HCC), human lung fibroblast cells (MRC-5), and macrophages. Cell culture media and cell images were collected, and matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor (VEGF) production was monitored under different cell culture conditions. We found that simulating hypoxia and/or serum starvation conditions induced elevated secretion of VEGF in the 3D co-culture model in vitro, but not MMP-1; the morphology of HCC in the 2D versus the 3D co-culture system was extremely different. MMP-1 and VEGF were secreted at higher levels in mixed cell groups rather than mono-culture groups. Therefore, incorporating lung cancer cells, fibroblasts, and macrophages may better reflect physiological metastasis mechanisms compared to mono-culture systems. Tumour stromal cells, macrophages, and fibroblast cells may promote invasion and metastasis, which also provides a new direction for the design of therapies targeted at destroying the stroma of tumor tissues. PMID:27232698

  2. Coaxial electrospray of liquid core-hydrogel shell microcapsules for encapsulation and miniaturized 3D culture of pluripotent stem cells

    PubMed Central

    Zhao, Shuting; Agarwal, Pranay; Rao, Wei; Huang, Haishui; Zhang, Renliang; Liu, Zhenguo; Yu, Jianhua; Weisleder, Noah; Zhang, Wujie; He, Xiaoming

    2014-01-01

    A novel coaxial electrospray technology is developed to generate microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells using two aqueous fluids in one step. Approximately 50 murine embryonic stem (ES) cells encapsulated in the core with high viability (92.3 ± 2.9%) can proliferate to form a single ES cell aggregate of 128.9 ± 17.4 μm in each microcapsule within 7 days. Quantitative analyses of gene and protein expression indicate that ES cells cultured in the miniaturized 3D liquid core of the core-shell microcapsules have significantly higher pluripotency on average than the cells cultured on 2D substrate or in the conventional 3D alginate hydrogel microbeads without a core-shell architecture. The higher pluripotency is further suggested by their significantly higher capability of differentiation into beating cardiomyocytes and higher expression of cardiomyocyte specific gene markers on average after directed differentiation under the same conditions. Considering its wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology together with the microcapsule system is of importance for mass production of ES cells with high pluripotency to facilitate translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic. PMID:25036382

  3. Influence of electrical stimulation on 3D-cultures of adipose tissue derived progenitor cells (ATDPCs) behavior.

    PubMed

    Castells-Sala, C; Sanchez, B; Recha-Sancho, L; Puig, V; Bragos, R; Semino, C E

    2012-01-01

    Tissue engineering has a fundamental role in regenerative medicine. Still today, the major motivation for cardiac regeneration is to design a platform that enables the complete tissue structure and physiological function regeneration of injured myocardium areas. Although tissue engineering approaches have been generally developed for two-dimensional (2D) culture systems, three-dimensional (3D) systems are being spotlighted as the means to mimic better in vivo cellular conditions. This manuscript examines the influence of electrical stimulation on 3D cultures of adipose tissue-derived progenitor cells (ATDPCs). ATDPCs cells were encapsulated into a self-assembling peptide nanoscaffold (RAD16-I) and continuously electro stimulated during 14-20 days with 2-ms pulses of 50mV/cm at a frequency of 1 Hz. Good cellular network formation and construct diameter reduction was observed in electro stimulated samples. Importantly, the process of electro stimulation does not disrupt cell viability or connectivity. As a future outlook, differentiation studies to cardiomyocytes-like cells will be performed analyzing gene profile and protein expression. PMID:23367213

  4. Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy

    PubMed Central

    Weber, Petra; Schickinger, Sarah; Wagner, Michael; Angres, Brigitte; Bruns, Thomas; Schneckenburger, Herbert

    2015-01-01

    Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure. PMID:25761242

  5. Accuracy of typical photogrammetric networks in cultural heritage 3D modeling projects

    NASA Astrophysics Data System (ADS)

    Nocerino, E.; Menna, F.; Remondino, F.

    2014-06-01

    The easy generation of 3D geometries (point clouds or polygonal models) with fully automated image-based methods poses nontrivial problems on how to check a posteriori the quality of the achieved results. Clear statements and procedures on how to plan the camera network, execute the survey and use automatic tools to achieve the prefixed requirements are still an open issue. Although such issues had been discussed and solved some years ago, the importance of camera network geometry is today often underestimated or neglected in the cultural heritage field. In this paper different camera network geometries, with normal and convergent images, are analyzed and the accuracy of the produced results are compared to ground truth measurements.

  6. Challenge for 3D culture technology: Application in carcinogenesis studies with human airway epithelial cells.

    PubMed

    Emura, M; Aufderheide, M

    2016-05-01

    Lung cancer is still one of the major intractable diseases and we urgently need more efficient preventive and curative measures. Recent molecular studies have provided strong evidence that allows us to believe that classically well-known early airway lesions such as hyperplasia, metaplasia, dysplasia and carcinoma in situ are really precancerous lesions progressing toward cancer but not necessarily transient and reversible alteration. This suggests that adequate early control of the precancerous lesions may lead to improved prevention of lung cancer. This knowledge is encouraging in view of the imminent necessity for additional experimental systems to investigate the causal mechanisms of cancers directly in human cells and tissues. There are many questions with regard to various precancerous lesions of the airways. For example, should cells, before reaching a stage of invasive carcinoma, undergo all precancerous stages such as hyperplasia or metaplasia and dysplasia, or is there any shortcut to bypass one or more of the precancerous stages? For the study of such questions, the emerging 3-dimensional (3D) cell culture technology appears to provide an effective and valuable tool. Though a great challenge, it is expected that this in vitro technology will be rapidly and reliably improved to enable the cultures to be maintained in an in vivo-mimicking state of differentiation for much longer than a period of at best a few months, as is currently the case. With the help of a "causes recombination-Lox" (Cre-lox) technology, it has been possible to trace cells giving rise to specific lung tumor types. In this short review we have attempted to assess the future role of 3D technology in the study of lung carcinogenesis. PMID:26951634

  7. Assessing Drug Efficacy in a Miniaturized Pancreatic Cancer In Vitro 3D Cell Culture Model.

    PubMed

    Shelper, Todd B; Lovitt, Carrie J; Avery, Vicky M

    2016-09-01

    Pancreatic cancer continues to have one of the poorest prognoses among all cancers. The drug discovery efforts for this disease have largely failed, with no significant improvement in survival outcomes for advanced pancreatic cancer patients over the past 20 years. Traditional in vitro cell culture techniques have been used extensively in both basic and early drug discovery; however, these systems offer poor models to assess emerging therapeutics. More predictive cell-based models, which better capture the cellular heterogeneity and complexities of solid pancreatic tumors, are urgently needed not only to improve drug discovery success but also to provide insight into the tumor biology. Pancreatic tumors are characterized by a unique micro-environment that is surrounded by a dense stroma. A complex network of interactions between extracellular matrix (ECM) components and the effects of cell-to-cell contacts may enhance survival pathways within in vivo tumors. This biological and physical complexity is lost in traditional cell monolayer models. To explore the predictive potential of a more complex cellular system, a three-dimensional (3D) micro-tumor assay was evaluated. Efficacy of six current chemotherapeutics was determined against a panel of primary and metastatic pancreatic tumor cell lines in a miniaturized ECM-based 3D cell culture system. Suitability for potential use in high-throughput screening applications was assessed, including ascertaining the effects that miniaturization and automation had on assay robustness. Cellular health was determined by utilizing an indirect population-based metabolic activity assay and a direct imaging-based cell viability assay. PMID:27552143

  8. Drug penetration and metabolism in 3D cell cultures treated in a 3D printed fluidic device: assessment of irinotecan via MALDI imaging mass spectrometry.

    PubMed

    LaBonia, Gabriel J; Lockwood, Sarah Y; Heller, Andrew A; Spence, Dana M; Hummon, Amanda B

    2016-06-01

    Realistic in vitro models are critical in the drug development process. In this study, a novel in vitro platform is employed to assess drug penetration and metabolism. This platform, which utilizes a 3D printed fluidic device, allows for dynamic dosing of three dimensional cell cultures, also known as spheroids. The penetration of the chemotherapeutic irinotecan into HCT 116 colon cancer spheroids was examined with MALDI imaging mass spectrometry (IMS). The active metabolite of irinotecan, SN-38, was also detected. After twenty-four hours of treatment, SN-38 was concentrated to the outside of the spheroid, a region of actively dividing cells. The irinotecan prodrug localization contrasted with SN-38 and was concentrated to the necrotic core of the spheroids, a region containing mostly dead and dying cells. These results demonstrate that this unique in vitro platform is an effective means to assess drug penetration and metabolism in 3D cell cultures. This innovative system can have a transformative impact on the preclinical evaluation of drug candidates due to its cost effectiveness and high throughput. PMID:27198560

  9. A 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials.

    PubMed

    Susewind, Julia; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Collnot, Eva-Maria; Schneider-Daum, Nicole; Griffiths, Gareth Wyn; Lehr, Claus-Michael

    2016-01-01

    Oral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO2, NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1β, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO2 or Au NPs. But once inflamed with IL-1β, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation. PMID:25738417

  10. Engineering a 3D microfluidic culture platform for tumor-treating field application

    PubMed Central

    Pavesi, Andrea; Adriani, Giulia; Tay, Andy; Warkiani, Majid Ebrahimi; Yeap, Wei Hseun; Wong, Siew Cheng; Kamm, Roger D.

    2016-01-01

    The limitations of current cancer therapies highlight the urgent need for a more effective therapeutic strategy. One promising approach uses an alternating electric field; however, the mechanisms involved in the disruption of the cancer cell cycle as well as the potential adverse effects on non-cancerous cells must be clarified. In this study, we present a novel microfluidic device with embedded electrodes that enables the application of an alternating electric field therapy to cancer cells in a 3D extracellular matrix. To demonstrate the potential of our system to aid in designing and testing new therapeutic approaches, cancer cells and cancer cell aggregates were cultured individually or co-cultured with endothelial cells. The metastatic potential of the cancer cells was reduced after electric field treatment. Moreover, the proliferation rate of the treated cancer cells was lower compared with that of the untreated cells, whereas the morphologies and proliferative capacities of the endothelial cells were not significantly affected. These results demonstrate that our novel system can be used to rapidly screen the effect of an alternating electric field on cancer and normal cells within an in vivo-like microenvironment with the potential to optimize treatment protocols and evaluate synergies between tumor-treating field treatment and chemotherapy. PMID:27215466

  11. Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture.

    PubMed

    DeJonge, Rachel E; Liu, Xiao-Ping; Deig, Christopher R; Heller, Stefan; Koehler, Karl R; Hashino, Eri

    2016-01-01

    Stem cell-derived inner ear sensory epithelia are a promising source of tissues for treating patients with hearing loss and dizziness. We recently demonstrated how to generate inner ear sensory epithelia, designated as inner ear organoids, from mouse embryonic stem cells (ESCs) in a self-organizing 3D culture. Here we improve the efficiency of this culture system by elucidating how Wnt signaling activity can drive the induction of otic tissue. We found that a carefully timed treatment with the potent Wnt agonist CHIR99021 promotes induction of otic vesicles-a process that was previously self-organized by unknown mechanisms. The resulting otic-like vesicles have a larger lumen size and contain a greater number of Pax8/Pax2-positive otic progenitor cells than organoids derived without the Wnt agonist. Additionally, these otic-like vesicles give rise to large inner ear organoids with hair cells whose morphological, biochemical and functional properties are indistinguishable from those of vestibular hair cells in the postnatal mouse inner ear. We conclude that Wnt signaling plays a similar role during inner ear organoid formation as it does during inner ear development in the embryo. PMID:27607106

  12. Engineering a 3D microfluidic culture platform for tumor-treating field application.

    PubMed

    Pavesi, Andrea; Adriani, Giulia; Tay, Andy; Warkiani, Majid Ebrahimi; Yeap, Wei Hseun; Wong, Siew Cheng; Kamm, Roger D

    2016-01-01

    The limitations of current cancer therapies highlight the urgent need for a more effective therapeutic strategy. One promising approach uses an alternating electric field; however, the mechanisms involved in the disruption of the cancer cell cycle as well as the potential adverse effects on non-cancerous cells must be clarified. In this study, we present a novel microfluidic device with embedded electrodes that enables the application of an alternating electric field therapy to cancer cells in a 3D extracellular matrix. To demonstrate the potential of our system to aid in designing and testing new therapeutic approaches, cancer cells and cancer cell aggregates were cultured individually or co-cultured with endothelial cells. The metastatic potential of the cancer cells was reduced after electric field treatment. Moreover, the proliferation rate of the treated cancer cells was lower compared with that of the untreated cells, whereas the morphologies and proliferative capacities of the endothelial cells were not significantly affected. These results demonstrate that our novel system can be used to rapidly screen the effect of an alternating electric field on cancer and normal cells within an in vivo-like microenvironment with the potential to optimize treatment protocols and evaluate synergies between tumor-treating field treatment and chemotherapy. PMID:27215466

  13. The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments

    PubMed Central

    Kanlaya, Rattiyaporn; Borkowski, Kamil; Schwämmle, Veit; Dai, Jie; Joensen, Kira Eyd; Wojdyla, Katarzyna; Carvalho, Vasco Botelho; Fey, Stephen J.

    2014-01-01

    Introduction Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Results Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. Summary We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance. PMID:25222612

  14. Engineering a 3D microfluidic culture platform for tumor-treating field application

    NASA Astrophysics Data System (ADS)

    Pavesi, Andrea; Adriani, Giulia; Tay, Andy; Warkiani, Majid Ebrahimi; Yeap, Wei Hseun; Wong, Siew Cheng; Kamm, Roger D.

    2016-05-01

    The limitations of current cancer therapies highlight the urgent need for a more effective therapeutic strategy. One promising approach uses an alternating electric field; however, the mechanisms involved in the disruption of the cancer cell cycle as well as the potential adverse effects on non-cancerous cells must be clarified. In this study, we present a novel microfluidic device with embedded electrodes that enables the application of an alternating electric field therapy to cancer cells in a 3D extracellular matrix. To demonstrate the potential of our system to aid in designing and testing new therapeutic approaches, cancer cells and cancer cell aggregates were cultured individually or co-cultured with endothelial cells. The metastatic potential of the cancer cells was reduced after electric field treatment. Moreover, the proliferation rate of the treated cancer cells was lower compared with that of the untreated cells, whereas the morphologies and proliferative capacities of the endothelial cells were not significantly affected. These results demonstrate that our novel system can be used to rapidly screen the effect of an alternating electric field on cancer and normal cells within an in vivo-like microenvironment with the potential to optimize treatment protocols and evaluate synergies between tumor-treating field treatment and chemotherapy.

  15. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    NASA Technical Reports Server (NTRS)

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  16. Mesoscale 3D manufacturing: varying focusing conditions for efficient direct laser writing of polymers

    NASA Astrophysics Data System (ADS)

    Jonušauskas, Linas; Malinauskas, Mangirdas

    2014-05-01

    In this paper, we report a novel approach for efficient fabrication of mesoscale polymer 3D microstructures. It is implemented by direct laser writing varying exposure beam focusing conditions. By carefully optimizing the fabrication parameters (laser intensity, scanning velocity/exposure time, changing objective lens) complex 3D geometries of the microstructures can be obtained rapidly. Additionally, we demonstrate this without the use of the photoinitiator as photosensitizer doped in the pre-polymer material (SZ2080). At femtosecond pulsed irradiation ~TW/cm² intensities the localized free radical polymerization is achieved via avalanche induced bond braking. Such microstructures have unique biocompatibility and optical transparency as well as optical damage threshold value. By creating the bulk part of the structure using low-NA (0.45) objective and subsequently fabricating the fine features using oil immersion high-NA (1.4) objective the manufacturing time is reduced dramatically (30x is demonstrated). Using this two objective method a prototype of functional microdevice was produced: 80 and 85 µm diameter microfluidic tubes with the fine filter consisting of 4 µm period grating structure that has 400 nm wide threads, which corresponds to a feature precision aspect ratio of ~200. Therefore, such method has great potential as a polymer fabrication tool for mesoscale optical, photonic and biomedical applications as well as highly integrated 3D µ-systems. Furthermore, the proposed approach is not limited to lithography and can be implemented in a more general type of laser writing, such as inscription within transparent materials or substractive manufacturing by ablation.

  17. Genotoxicity assessment of reactive and disperse textile dyes using human dermal equivalent (3D cell culture system).

    PubMed

    Leme, Daniela Morais; Primo, Fernando Lucas; Gobo, Graciely Gomides; da Costa, Cleber Rafael Vieira; Tedesco, Antonio Claudio; de Oliveira, Danielle Palma

    2015-01-01

    Thousands of dyes are marketed daily for different purposes, including textile dyeing. However, there are several studies reporting attributing to dyes deleterious human effects such as DNA damage. Humans may be exposed to toxic dyes through either ingestion of contaminated waters or dermal contact with colored garments. With respect to dermal exposure, human skin equivalents are promising tools to assess in vitro genotoxicity of dermally applied chemicals using a three-dimensional (3D) model to mimic tissue behavior. This study investigated the sensitivity of an in-house human dermal equivalent (DE) for detecting genotoxicity of textile dyes. Two azo (reactive green 19 [RG19] and disperse red 1[DR1]) dyes and one anthraquinone (reactive blue 2 [RB2]) dye were analyzed. RG19 was genotoxic for DE in a dose-responsive manner, whereas RB2 and DR1 were nongenotoxic under the conditions tested. These findings are not in agreement with previous genotoxicological assessment of these dyes carried out using two-dimensional (2D) cell cultures, which showed that DR1 was genotoxic in human hepatoma cells (HepG2) and RG19 was nongenotoxic for normal human dermal fibroblasts (NHDF). These discrepant results probably may be due to differences between metabolic activities of each cell type (organ-specific genotoxicity, HepG2 and fibroblasts) and the test setup systems used in each study (fibroblasts cultured at 2D and three-dimensional [3D] culture systems). Genotoxicological assessment of textile dyes in context of organ-specific genotoxicity and using in vitro models that more closely resemble in vivo tissue architecture and physiology may provide more reliable estimates of genotoxic potential of these chemicals. PMID:25785560

  18. Effect of Lyso-phosphatidylcholine and Schnurri-3 on Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells to Calcifying Vascular Cells in 3D Culture

    PubMed Central

    Castro-Chavez, Fernando; Vickers, Kasey C.; Sam Lee, Jae; Tung, Ching-Hsuan; Morrisett, Joel D.

    2015-01-01

    Background In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell mono-layers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo. Methods and results To simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold–polyvmer–iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3–4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10 nM lysophosphatidylcholine (LPC), for 3 days, cell clusters exhibited translucent extensions/rods 60–80 µm wide and 200–250 µm long. When 0.5 µg/µl Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590–650 nm light, these extensions emitted intrinsic fluorescence at >667 nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518 nm, the λmax for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminogly-cans than cells treated with LPC and Schnurri-3. Conclusions In 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3. General significance The formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed

  19. Radiation Quality Effects on Transcriptome Profiles in 3-d Cultures After Particle Irradiation

    NASA Technical Reports Server (NTRS)

    Patel, Z. S.; Kidane, Y. H.; Huff, J. L.

    2014-01-01

    In this work, we evaluate the differential effects of low- and high-LET radiation on 3-D organotypic cultures in order to investigate radiation quality impacts on gene expression and cellular responses. Reducing uncertainties in current risk models requires new knowledge on the fundamental differences in biological responses (the so-called radiation quality effects) triggered by heavy ion particle radiation versus low-LET radiation associated with Earth-based exposures. We are utilizing novel 3-D organotypic human tissue models that provide a format for study of human cells within a realistic tissue framework, thereby bridging the gap between 2-D monolayer culture and animal models for risk extrapolation to humans. To identify biological pathway signatures unique to heavy ion particle exposure, functional gene set enrichment analysis (GSEA) was used with whole transcriptome profiling. GSEA has been used extensively as a method to garner biological information in a variety of model systems but has not been commonly used to analyze radiation effects. It is a powerful approach for assessing the functional significance of radiation quality-dependent changes from datasets where the changes are subtle but broad, and where single gene based analysis using rankings of fold-change may not reveal important biological information. We identified 45 statistically significant gene sets at 0.05 q-value cutoff, including 14 gene sets common to gamma and titanium irradiation, 19 gene sets specific to gamma irradiation, and 12 titanium-specific gene sets. Common gene sets largely align with DNA damage, cell cycle, early immune response, and inflammatory cytokine pathway activation. The top gene set enriched for the gamma- and titanium-irradiated samples involved KRAS pathway activation and genes activated in TNF-treated cells, respectively. Another difference noted for the high-LET samples was an apparent enrichment in gene sets involved in cycle cycle/mitotic control. It is

  20. Critical analysis of 3-D organoid in vitro cell culture models for high-throughput drug candidate toxicity assessments.

    PubMed

    Astashkina, Anna; Grainger, David W

    2014-04-01

    Drug failure due to toxicity indicators remains among the primary reasons for staggering drug attrition rates during clinical studies and post-marketing surveillance. Broader validation and use of next-generation 3-D improved cell culture models are expected to improve predictive power and effectiveness of drug toxicological predictions. However, after decades of promising research significant gaps remain in our collective ability to extract quality human toxicity information from in vitro data using 3-D cell and tissue models. Issues, challenges and future directions for the field to improve drug assay predictive power and reliability of 3-D models are reviewed. PMID:24613390

  1. Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices.

    PubMed

    Casey, A; Gargotti, M; Bonnier, F; Byrne, H J

    2016-06-01

    The interest in the use of 3D matrices for in vitro analysis, with a view to increasing the relevance of in vitro studies and reducing the dependence on in vivo studies, has been growing in recent years. Cells grown in a 3D in vitro matrix environment have been reported to exhibit significantly different properties to those in a conventional 2D culture environment. However, comparison of 2D and 3D cell culture models have recently been noted to result in differing responses of cytotoxic assays, without any associated change in viability. The effect was attributed to differing conversion rates and effective concentrations of the resazurin assay in 2D and 3D environments, rather than differences in cellular metabolism. In this study, the efficacy of a chemotherapeutic agent, doxorubicin, is monitored and compared in conventional 2D and 3D collagen gel exposures of immortalized human cervical cells. Viability was monitored with the aid of the Alamar Blue assay and drug internalisation was verified using confocal microscopy. Drug uptake and retention within the collagen matrix was monitored by absorption spectroscopy. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to a 3D environment causing alterations to dye resazurin uptake and conversion rates. The use of 3D culture matrices has widely been interpreted to result in "reduced" toxicity or cellular "resistance" to the chemotherapeutic agent. The results of this study show that the reduced efficiency of the drug to cells grown in the 3D environment can be accounted for by a sequential reduction of the effective concentration of the test compound and assay. This is due to absorption within the collagen gel inducing a higher uptake of both drug and assay thereby influencing the toxic impact of the drug and conversion rate of resazurin, and. The increased effective surface area of the cell exposed to the drug

  2. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

    NASA Astrophysics Data System (ADS)

    Lei, Yuguo; Schaffer, David V.

    2013-12-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

  3. Bioinspired Tuning of Hydrogel Permeability-Rigidity Dependency for 3D Cell Culture

    NASA Astrophysics Data System (ADS)

    Lee, Min Kyung; Rich, Max H.; Baek, Kwanghyun; Lee, Jonghwi; Kong, Hyunjoon

    2015-03-01

    Hydrogels are being extensively used for three-dimensional immobilization and culture of cells in fundamental biological studies, biochemical processes, and clinical treatments. However, it is still a challenge to support viability and regulate phenotypic activities of cells in a structurally stable gel, because the gel becomes less permeable with increasing rigidity. To resolve this challenge, this study demonstrates a unique method to enhance the permeability of a cell-laden hydrogel while avoiding a significant change in rigidity of the gel. Inspired by the grooved skin textures of marine organisms, a hydrogel is assembled to present computationally optimized micro-sized grooves on the surface. Separately, a gel is engineered to preset aligned microchannels similar to a plant's vascular bundles through a uniaxial freeze-drying process. The resulting gel displays significantly increased water diffusivity with reduced changes of gel stiffness, exclusively when the microgrooves and microchannels are aligned together. No significant enhancement of rehydration is achieved when the microgrooves and microchannels are not aligned. Such material design greatly enhances viability and neural differentiation of stem cells and 3D neural network formation within the gel.

  4. Curved and folded micropatterns in 3D cell culture and tissue engineering.

    PubMed

    Yilmaz, Cem Onat; Xu, Zinnia S; Gracias, David H

    2014-01-01

    Cells live in a highly curved and folded micropatterned environment within the human body. Hence, there is a need to develop engineering paradigms to replicate these microenvironments in order to investigate the behavior of cells in vitro, as well as to develop bioartificial organs for tissue engineering and regenerative medicine. In this chapter, we first motivate the need for such micropatterns based on anatomical considerations and then survey methods that can be utilized to generate curved and folded micropatterns of relevance to 3D cell culture and tissue engineering. The methods surveyed can broadly be divided into two classes: top-down approaches inspired by conventional 2D microfabrication and bottom-up approaches most notably in the self-assembly of thin patterned films. These methods provide proof of concept that the high resolution, precise and reproducible patterning of cell and matrix microenvironments in anatomically relevant curved and folded geometries is possible. A specific protocol is presented to create curved and folded hydrogel micropatterns. PMID:24560507

  5. Accuracy of cultural heritage 3D models by RPAS and terrestrial photogrammetry

    NASA Astrophysics Data System (ADS)

    Bolognesi, M.; Furini, A.; Russo, V.; Pellegrinelli, A.; Russo, P.

    2014-06-01

    The combined use of high-resolution digital images taken from ground as well as from RPAS (Remotely Piloted Aircraft Systems) have significantly increased the potential of close range digital photogrammetry applications in Cultural Heritage surveying and modeling. It is in fact possible, thanks to SfM (Structure from Motion), to simultaneously process great numbers of aerial and terrestrial images for the production of a dense point cloud of an object. In order to analyze the accuracy of results, we started numerous tests based on the comparison between 3D digital models of a monumental complex realized by the integration of aerial and terrestrial photogrammetry and an accurate TLS (Terrestrial Laser Scanner) reference model of the same object. A lot of digital images of a renaissance castle, assumed as test site, have been taken both by ground level and by RPAS at different distances and flight altitudes and with different flight patterns. As first step of the experimentation, the images were previously processed with Agisoft PhotoScan, one of the most popular photogrammetric software. The comparison between the photogrammetric DSM of the monument and a TLS reference one was carried out by evaluating the average deviation between the points belonging to the two entities, both globally and locally, on individual façades and architectural elements (sections and particular). In this paper the results of the first test are presented. A good agreement between photogrammetric and TLS digital models of the castle is pointed out.

  6. Bioinspired Tuning of Hydrogel Permeability-Rigidity Dependency for 3D Cell Culture

    PubMed Central

    Lee, Min Kyung; Rich, Max H.; Baek, Kwanghyun; Lee, Jonghwi; Kong, Hyunjoon

    2015-01-01

    Hydrogels are being extensively used for three-dimensional immobilization and culture of cells in fundamental biological studies, biochemical processes, and clinical treatments. However, it is still a challenge to support viability and regulate phenotypic activities of cells in a structurally stable gel, because the gel becomes less permeable with increasing rigidity. To resolve this challenge, this study demonstrates a unique method to enhance the permeability of a cell-laden hydrogel while avoiding a significant change in rigidity of the gel. Inspired by the grooved skin textures of marine organisms, a hydrogel is assembled to present computationally optimized micro-sized grooves on the surface. Separately, a gel is engineered to preset aligned microchannels similar to a plant's vascular bundles through a uniaxial freeze-drying process. The resulting gel displays significantly increased water diffusivity with reduced changes of gel stiffness, exclusively when the microgrooves and microchannels are aligned together. No significant enhancement of rehydration is achieved when the microgrooves and microchannels are not aligned. Such material design greatly enhances viability and neural differentiation of stem cells and 3D neural network formation within the gel. PMID:25752700

  7. Effect of particle size in a colloidal hydrogel scaffold for 3D cell culture.

    PubMed

    Gu, Jianjun; Zhao, Yening; Guan, Ying; Zhang, Yongjun

    2015-12-01

    The in situ-forming colloidal hydrogels from the thermal gelation of poly(N-isopropylacrylamide) (PNIPAM) microgel dispersions have been exploited for 3D cell culture. The properties of the hydrogel scaffold need to be tuned to further improve its performance. In addition, cellular uptake of the microgel particles need to be reduced to avoid their potential undesired influence. For these purposes we systematically examined the effect of microgel particle size on the hydrogel scaffold. It was found that gel properties could be tuned via changing particle size. Increasing particle size reduces the gel strength and its syneresis degree, both of which are favorable for cell growth. Meanwhile increasing particle size could also reduce significantly the cellular uptake of the microgel particles. Microgel with a size of ~162 nm shows the highest cellular uptake, beyond which cellular uptake decreases with increasing particle size. Hydrogel scaffold from 300 nm microgel, with suitable physical properties and reduced cellular uptake, were successfully used for multicellular spheroid generation. PMID:26613865

  8. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

    NASA Technical Reports Server (NTRS)

    Bancroft, Gregory N.; Sikavitsas, Vassilios I.; van den Dolder, Juliette; Sheffield, Tiffany L.; Ambrose, Catherine G.; Jansen, John A.; Mikos, Antonios G.; McIntire, L. V. (Principal Investigator)

    2002-01-01

    Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

  9. Potential and limitations of microscopy and Raman spectroscopy for live-cell analysis of 3D cell cultures.

    PubMed

    Charwat, Verena; Schütze, Karin; Holnthoner, Wolfgang; Lavrentieva, Antonina; Gangnus, Rainer; Hofbauer, Pablo; Hoffmann, Claudia; Angres, Brigitte; Kasper, Cornelia

    2015-07-10

    Today highly complex 3D cell culture formats that closely mimic the in vivo situation are increasingly available. Despite their wide use, the development of analytical methods and tools that can work within the depth of 3D-tissue constructs lags behind. In order to get the most information from a 3D cell sample, adequate and reliable assays are required. However, the majority of tools and methods used today have been originally designed for 2D cell cultures and translation to a 3D environment is in general not trivial. Ideally, an analytical method should be non-invasive and allow for repeated observation of living cells in order to detect dynamic changes in individual cells within the 3D cell culture. Although well-established laser confocal microscopy can be used for these purposes, this technique has serious limitations including penetration depth and availability. Focusing on two relevant analytical methods for live-cell monitoring, we discuss the current challenges of analyzing living 3D samples: microscopy, which is the most widely used technology to observe and examine cell cultures, has been successfully adapted for 3D samples by recording of so-called "z-stacks". However the required equipment is generally very expensive and therefore access is often limited. Consequently alternative and less advanced approaches are often applied that cannot capture the full structural complexity of a 3D sample. Similarly, image analysis tools for quantification of microscopic images range from highly specialized and costly to simplified and inexpensive. Depending on the actual sample composition and scientific question the best approach needs to be assessed individually. Another more recently introduced technology for non-invasive cell analysis is Raman micro-spectroscopy. It enables label-free identification of cellular metabolic changes with high sensitivity and has already been successful applied to 2D and 3D cell cultures. However, its future significance for cell

  10. A 3D sphere culture system containing functional polymers for large-scale human pluripotent stem cell production.

    PubMed

    Otsuji, Tomomi G; Bin, Jiang; Yoshimura, Azumi; Tomura, Misayo; Tateyama, Daiki; Minami, Itsunari; Yoshikawa, Yoshihiro; Aiba, Kazuhiro; Heuser, John E; Nishino, Taito; Hasegawa, Kouichi; Nakatsuji, Norio

    2014-05-01

    Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production. PMID:24936458

  11. Comparison of 3d Reconstruction Services and Terrestrial Laser Scanning for Cultural Heritage Documentation

    NASA Astrophysics Data System (ADS)

    Rasztovits, S.; Dorninger, P.

    2013-07-01

    Terrestrial Laser Scanning (TLS) is an established method to reconstruct the geometrical surface of given objects. Current systems allow for fast and efficient determination of 3D models with high accuracy and richness in detail. Alternatively, 3D reconstruction services are using images to reconstruct the surface of an object. While the instrumental expenses for laser scanning systems are high, upcoming free software services as well as open source software packages enable the generation of 3D models using digital consumer cameras. In addition, processing TLS data still requires an experienced user while recent web-services operate completely automatically. An indisputable advantage of image based 3D modeling is its implicit capability for model texturing. However, the achievable accuracy and resolution of the 3D models is lower than those of laser scanning data. Within this contribution, we investigate the results of automated web-services for image based 3D model generation with respect to a TLS reference model. For this, a copper sculpture was acquired using a laser scanner and using image series of different digital cameras. Two different webservices, namely Arc3D and AutoDesk 123D Catch were used to process the image data. The geometric accuracy was compared for the entire model and for some highly structured details. The results are presented and interpreted based on difference models. Finally, an economical comparison of the generation of the models is given considering the interactive and processing time costs.

  12. Differential effects of MAPK pathway inhibitors on migration and invasiveness of BRAF(V600E) mutant thyroid cancer cells in 2D and 3D culture.

    PubMed

    Ingeson-Carlsson, Camilla; Martinez-Monleon, Angela; Nilsson, Mikael

    2015-11-01

    Tumor microenvironment influences targeted drug therapy. In this study we compared drug responses to RAF and MEK inhibitors on tumor cell migration in 2D and 3D culture of BRAF(V600E) mutant cell lines derived from human papillary (BCPAP) and anaplastic (SW1736) thyroid carcinomas. Scratch wounding was compared to a double-layered collagen gel model developed for analysis of directed tumor cell invasion during prolonged culture. In BCPAP both PLX4720 and U0126 inhibited growth and migration in 2D and decreased tumor cell survival in 3D. In SW1736 drugs had no effect on migration in 2D but decreased invasion in 3D, however this related to reduced growth. Dual inhibition of BRAF(V600E) and MEK reduced but did not prevent SW1736 invasion although rebound phosphorylation of ERK in response to PLX4720 was blocked by U0126. These findings indicate that anti-tumor drug effects in vitro differ depending on culture conditions (2D vs. 3D) and that the invasive features of anaplastic thyroid cancer depend on non-MEK mechanism(s). PMID:26384551

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  15. Carbon deposition from aromatic solvents onto active intact 3d metal surface at ambient conditions.

    PubMed

    Safronov, A P; Kurlyandskaya, G V; Chlenova, A A; Kuznetsov, M V; Bazhin, D N; Beketov, I V; Sanchez-Ilarduya, M B; Martinez-Amesti, A

    2014-03-25

    The process of carbon deposition onto 3d metal surface immersed in aromatic solvents (benzene, toluene, xylene) at ambient conditions was studied for as-prepared magnetic nanoparticles (MNPs) and Fe-based films by thermal analysis, mass spectrometry, X-ray diffraction, X-ray photoelectron spectroscopy, Raman spectroscopy, electron microscopy, and energy-dispersive X-ray spectroscopy. The mechanism of the deposition at the interface is likely the heterogeneous Scholl oxidation of the aromatic hydrocarbons, which is the cationic polymerization of the aryl rings. It results in the formation of polycyclic aromatic hydrocarbons (PAH) chemically bonded to the surface of a MNP or thin metallic film. The benzene rings in the polycyclic deposit do not maintain planar aligned structures and do not provide delocalization of the π-electrons in the zone structure. Contrary to the dense graphite layers, the polycyclic layers, although chemically bonded, are not attached tightly to the surface. Such "hairlike" structure of the carboneous deposit might be especially favorable for the applications that imply the enhanced interaction at the surfaces incorporated in the functional matrices (polymeric composites or biosensors). The aromatic chemical nature of the deposit provides strong interaction with most polymers, while its loose structure favors conformational mobility of macromolecular chains at the interface. PMID:24593324

  16. 3-D simulation of gases transport under condition of inert gas injection into goaf

    NASA Astrophysics Data System (ADS)

    Liu, Mao-Xi; Shi, Guo-Qing; Guo, Zhixiong; Wang, Yan-Ming; Ma, Li-Yang

    2016-02-01

    To prevent coal spontaneous combustion in mines, it is paramount to understand O2 gas distribution under condition of inert gas injection into goaf. In this study, the goaf was modeled as a 3-D porous medium based on stress distribution. The variation of O2 distribution influenced by CO2 or N2 injection was simulated based on the multi-component gases transport and the Navier-Stokes equations using Fluent. The numerical results without inert gas injection were compared with field measurements to validate the simulation model. Simulations with inert gas injection show that CO2 gas mainly accumulates at the goaf floor level; however, a notable portion of N2 gas moves upward. The evolution of the spontaneous combustion risky zone with continuous inert gas injection can be classified into three phases: slow inerting phase, rapid accelerating inerting phase, and stable inerting phase. The asphyxia zone with CO2 injection is about 1.25-2.4 times larger than that with N2 injection. The efficacy of preventing and putting out mine fires is strongly related with the inert gas injecting position. Ideal injections are located in the oxidation zone or the transitional zone between oxidation zone and heat dissipation zone.

  17. Infrared imaging of MDA-MB-231 breast cancer cell line phenotypes in 2D and 3D cultures.

    PubMed

    Smolina, Margarita; Goormaghtigh, Erik

    2015-04-01

    One current challenge in the field of breast cancer infrared imaging is the identification of carcinoma cell subtypes in the tissue. Neither sequencing nor immunochemistry is currently able to provide a cell by cell thorough classification. The latter is needed to build accurate statistical models capable of recognizing the diversity of breast cancer cell lines that may be present in a tissue section. One possible approach for overcoming this problem is to obtain the IR spectral signature of well-characterized tumor cell lines in culture. Cultures in three-dimensional matrices appear to generate an environment that mimics better the in vivo environment. There are, at present, series of breast cancer cell lines that have been thoroughly characterized in two- and three-dimensional (2D and 3D) cultures by full transcriptomics analyses. In this work, we describe the methods used to grow, to process, and to characterize a triple-negative breast cancer cell line, MDA-MB-231, in 3D laminin-rich extracellular matrix (lrECM) culture and compare it with traditional monolayer cultures and tissue sections. While unsupervised analyses did not completely separate spectra of cells grown in 2D from 3D lrECM cultures, a supervised statistical analysis resulted in an almost perfect separation. When IR spectral responses of epithelial tumor cells from clinical triple-negative breast carcinoma samples were added to these data, a principal component analysis indicated that they cluster closer to the spectra of 3D culture cells than to the spectra of cells grown on a flat plastic substrata. This result is encouraging because of correlating well-characterized cell line features with clinical biopsies. PMID:25568895

  18. Sensitivity of an asymmetric 3D diffuser to vortex-generator induced inlet condition perturbations

    NASA Astrophysics Data System (ADS)

    Grundmann, S.; Sayles, E. L.; Elkins, Christopher J.; Eaton, J. K.

    2012-01-01

    Modifications of the turbulent separated flow in an asymmetric three-dimensional diffuser due to inlet condition perturbations were investigated using conventional static pressure measurements and velocity data acquired using magnetic resonance velocimetry (MRV). Previous experiments and simulations revealed a strong sensitivity of the diffuser performance to weak secondary flows in the inlet. The present, more detailed experiments were conducted to obtain a better understanding of this sensitivity. Pressure data were acquired in an airflow apparatus at an inlet Reynolds number of 10,000. The diffuser pressure recovery was strongly affected by a pair of longitudinal vortices injected along one wall of the inlet channel using either dielectric barrier discharge plasma actuators or conventional half-delta wing vortex generators. MRV measurements were obtained in a water flow apparatus at matched Reynolds number for two different cases with passive vortex generators. The first case had a pair of counter-rotating longitudinal vortices embedded in the boundary layer near the center of the expanding wall of the diffuser such that the flow on the outsides of the vortices was directed toward the wall. The MRV data showed that the three-dimensional separation bubble initially grew much slower causing a rapid early reduction in the core flow velocity and a consequent reduction of total pressure losses due to turbulent mixing. This produced a 13% increase in the overall pressure recovery. For the second case, the vortices rotated in the opposite sense, and the image vortices pushed them into the corners. This led to a very rapid initial growth of the separation bubble and formation of strong swirl at the diffuser exit. These changes resulted in a 17% reduction in the overall pressure recovery for this case. The results emphasize the extreme sensitivity of 3D separated flows to weak perturbations.

  19. State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

    PubMed

    Alépée, Natalie; Bahinski, Anthony; Daneshian, Mardas; De Wever, Bart; Fritsche, Ellen; Goldberg, Alan; Hansmann, Jan; Hartung, Thomas; Haycock, John; Hogberg, Helena; Hoelting, Lisa; Kelm, Jens M; Kadereit, Suzanne; McVey, Emily; Landsiedel, Robert; Leist, Marcel; Lübberstedt, Marc; Noor, Fozia; Pellevoisin, Christian; Petersohn, Dirk; Pfannenbecker, Uwe; Reisinger, Kerstin; Ramirez, Tzutzuy; Rothen-Rutishauser, Barbara; Schäfer-Korting, Monika; Zeilinger, Katrin; Zurich, Marie-Gabriele

    2014-01-01

    Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing. PMID:25027500

  20. 3D Visualization of Cultural Heritage Artefacts with Virtual Reality devices

    NASA Astrophysics Data System (ADS)

    Gonizzi Barsanti, S.; Caruso, G.; Micoli, L. L.; Covarrubias Rodriguez, M.; Guidi, G.

    2015-08-01

    Although 3D models are useful to preserve the information about historical artefacts, the potential of these digital contents are not fully accomplished until they are not used to interactively communicate their significance to non-specialists. Starting from this consideration, a new way to provide museum visitors with more information was investigated. The research is aimed at valorising and making more accessible the Egyptian funeral objects exhibited in the Sforza Castle in Milan. The results of the research will be used for the renewal of the current exhibition, at the Archaeological Museum in Milan, by making it more attractive. A 3D virtual interactive scenario regarding the "path of the dead", an important ritual in ancient Egypt, was realized to augment the experience and the comprehension of the public through interactivity. Four important artefacts were considered for this scope: two ushabty, a wooden sarcophagus and a heart scarab. The scenario was realized by integrating low-cost Virtual Reality technologies, as the Oculus Rift DK2 and the Leap Motion controller, and implementing a specific software by using Unity. The 3D models were implemented by adding responsive points of interest in relation to important symbols or features of the artefact. This allows highlighting single parts of the artefact in order to better identify the hieroglyphs and provide their translation. The paper describes the process for optimizing the 3D models, the implementation of the interactive scenario and the results of some test that have been carried out in the lab.

  1. An appropriate selection of a 3D alginate culture model for hepatic Huh-7 cell line encapsulation intended for viral studies.

    PubMed

    Tran, Nhu Mai; Dufresne, Murielle; Duverlie, Gilles; Castelain, Sandrine; Défarge, Christian; Paullier, Patrick; Legallais, Cecile

    2013-01-01

    Three-dimensional (3D) culture systems have been introduced to provide cells with a biomimetic environment that is similar to in vivo conditions. Among the polymeric molecules available, sodium-alginate (Na-alg) salt is a material that is currently employed in different areas of drug delivery and tissue engineering, because it offers biocompatibility and optimal chemical properties, and its gelation with calcium chloride provides calcium-alginate (Ca-alg) scaffolds with mechanical stability and relative permeability. In this work, four different preparations of Ca-alg beads with varying Na-alg viscosity and concentration were used for a human hepatoma cell line (Huh-7) encapsulation. The effects of Ca-alg bead preparation on structural cell organization, liver-specific functions, and the expression of specific receptors implicated in hepatotropic virus permissivity were evaluated. Hepatic cells were cultured in 500 μm diameter Ca-alg beads for 7 days under dynamic conditions. For all culture systems, cell viability reached almost 100% at day 7. Cell proliferation was concomitantly followed by hepatocyte organization in aggregates, which adopted two different morphologies (spheroid aggregates or multicellular channel-like structures), depending on Ca-alg bead preparation. These cellular organizations established a real 3D hepatocyte architecture with cell polarity, cell junctions, and abundant bile canaliculi possessing microvillus-lined channels. The functionality of these 3D cultures was confirmed by the production of albumin and the exhibition of CYP1A activity over culture time, which were variable, according to Ca-alg bead condition. The expression of specific receptors of hepatitis C virus by Huh-7 cells suggests encouraging data for the further development of a new viral culture system in Ca-alg beads. In summary, this 3D hepatic cell culture represents a promising physiologically relevant system for further in vitro studies and demonstrates that an

  2. Assessments for 3d Reconstructions of Cultural Heritage Using Digital Technologies

    NASA Astrophysics Data System (ADS)

    Manferdini, A. M.; Galassi, M.

    2013-02-01

    The aim of this contribution is to show the results of evaluations on 3D digitizations performed using different methodologies and technologies. In particular, for surveys conducted at the architectural and urban scale, the recent reduction of costs related to Time of Flight and phase shift laser scanners is actually enhancing the replacement of traditional topographic instruments (i.e. total stations) with range-based technologies for the acquisition of 3D data related to built heritage. If compared to surveys performed using traditional topographic technologies, range-based ones offer a wide range of advantages, but they also require different skills, procedures and times. The present contribution shows the results of a practical application of both approaches on the same case study. Another application was suggested by the recent developments in the photogrammetric field that enhance the improvement of software able to automatically orient uncalibrated cameras and derive dense and accurate 3D point clouds, with evident benefits in reduction of costs required for survey equipment. Therefore, the presented case study constituted the occasion to compare a rangebased survey with a fast 3D acquisition and modelling using a Structure from Motion solution. These survey procedures were adopted at an architectural scale, on a single building, that was surveyed both on the outside and on the inside. Assessments on the quality of the rebuilt information is reported, as far as metric accuracy and reliability is concerned, as well as on time consuming and on skills required during each step of the adopted pipelines. For all approaches, these analysis highlighted advantages and disadvantages that allow to conduct evaluations on the possible convenience of adopting range-based technologies instead of a traditional topographic approach or a photogrammetric one instead of a range based one in case of surveys conducted at an architectural/urban scale.

  3. A microfabricated magnetic actuation device for mechanical conditioning of arrays of 3D microtissues.

    PubMed

    Xu, Fan; Zhao, Ruogang; Liu, Alan S; Metz, Tristin; Shi, Yu; Bose, Prasenjit; Reich, Daniel H

    2015-06-01

    This paper describes an approach to actuate magnetically arrays of microtissue constructs for long-term mechanical conditioning and subsequent biomechanical measurements. Each construct consists of cell/matrix material self-assembled around a pair of flexible poly(dimethylsiloxane) (PDMS) pillars. The deflection of the pillars reports the tissues' contractility. Magnetic stretching of individual microtissues via magnetic microspheres mounted on the cantilevers has been used to elucidate the tissues' elastic modulus and response to varying mechanical boundary conditions. This paper describes the fabrication of arrays of micromagnetic structures that can transduce an externally applied uniform magnetic field to actuate simultaneously multiple microtissues. These structures are fabricated on silicon-nitride coated Si wafers and contain electrodeposited Ni bars. Through-etched holes provide optical and culture media access when the devices are mounted on the PDMS microtissue scaffold devices. Both static and AC forces (up to 20 μN on each microtissue) at physiological frequencies are readily generated in external fields of 40 mT. Operation of the magnetic arrays was demonstrated via measurements of elastic modulus and dynamic stiffening in response to AC actuation of fibroblast populated collagen microtissues. PMID:25959132

  4. A microfabricated magnetic actuation device for mechanical conditioning of arrays of 3D microtissues

    PubMed Central

    Xu, Fan; Zhao, Ruogang; Liu, Alan S.; Metz, Tristin; Shi, Yu; Bose, Prasenjit; Reich, Daniel H.

    2015-01-01

    This paper describes an approach to actuate magnetically arrays of microtissue constructs for long-term mechanical conditioning and subsequent biomechanical measurements. Each construct consists of cell/matrix material self-assembled around a pair of flexible poly(dimethylsiloxane) (PDMS) pillars. The deflection of the pillars reports the tissues’ contractility. Magnetic stretching of individual microtissues via magnetic microspheres mounted on the cantilevers has been used to elucidate the tissues’ elastic modulus and response to varying mechanical boundary conditions. This paper describes the fabrication of arrays of micromagnetic structures that can transduce an externally applied uniform magnetic field to actuate simultaneously multiple microtissues. These structures are fabricated on silicon-nitride coated Si wafers and contain electrodeposited Ni bars. Through-etched holes provide optical and culture media access when the devices are mounted on the PDMS microtissue scaffold devices. Both static and AC forces (up to 20 μN on each microtissue) at physiological frequencies are readily generated in external fields of 40 mT. Operation of the magnetic arrays was demonstrated via measurements of elastic modulus and dynamic stiffening in response to AC actuation of fibroblast populated collagen microtissues. PMID:25959132

  5. Mechanisms of DNA damage response to targeted irradiation in organotypic 3D skin cultures.

    PubMed

    Acheva, Anna; Ghita, Mihaela; Patel, Gaurang; Prise, Kevin M; Schettino, Giuseppe

    2014-01-01

    DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2. PMID:24505255

  6. Implementation of wall boundary conditions for transpiration in F3D thin-layer Navier-Stokes code

    NASA Technical Reports Server (NTRS)

    Kandula, M.; Martin, F. W., Jr.

    1991-01-01

    Numerical boundary conditions for mass injection/suction at the wall are incorporated in the thin-layer Navier-Stokes code, F3D. The accuracy of the boundary conditions and the code is assessed by a detailed comparison of the predictions of velocity distributions and skin-friction coefficients with exact similarity solutions for laminar flow over a flat plate with variable blowing/suction, and measurements for turbulent flow past a flat plate with uniform blowing. In laminar flow, F3D predictions for friction coefficient compare well with exact similarity solution with and without suction, but produces large errors at moderate-to-large values of blowing. A slight Mach number dependence of skin-friction coefficient due to blowing in turbulent flow is computed by F3D code. Predicted surface pressures for turbulent flow past an airfoil with mass injection are in qualitative agreement with measurements for a flat plate.

  7. Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

    PubMed Central

    Zhou, Hong; Malik, Malika Amattullah; Arab, Aarthi; Hill, Matthew Thomas; Shikanov, Ariella

    2015-01-01

    Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in

  8. Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles.

    PubMed

    Zhou, Hong; Malik, Malika Amattullah; Arab, Aarthi; Hill, Matthew Thomas; Shikanov, Ariella

    2015-01-01

    Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in

  9. Simulation of 3D effects on partially detached divertor conditions in NSTX and Alcator C-Mod

    NASA Astrophysics Data System (ADS)

    Lore, Jeremy

    2014-10-01

    Establishing a validated, predictive capability for the divertor plasma is critical for future fusion reactors, which must operate with detached divertors to reduce peak heat fluxes to the plasma facing components (PFC) and to mitigate net material erosion. This is challenging even for existing 2D codes, and is complicated further by non-axisymmetric effects due to divertor-localized gas injection, 3D magnetic fields, and 3D PFCs, modeling of which requires 3D simulations. New experiments performed on C-Mod at the request of the ITER organization to examine the consequence of localized nitrogen gas injection, show clear toroidal asymmetries in radiated power, impurity radiation, and divertor pressure. The 3D plasma/neutral transport code EMC3-EIRENE has been applied to model these experiments in the first attempt to benchmark the code against tokamak experimental data under detached conditions. The measured pressure modulation and the impurity radiation trends in the edge are qualitatively reproduced by the simulations, which also predict a ~2x modulation in heat flux at the outer strike point. Discrepancies are found in comparison with the measured private region radiation, and the simulations also indicate colder, denser divertor conditions than measured, suggesting that drifts and kinetic corrections may be required for more quantitative agreement. In separate experiments on NSTX, detached divertor plasmas are observed to reattach when 3D fields are applied. Modeling of the NSTX experiments reproduces these trends, with an increased peak heat flux with 3D fields and qualitative agreement of the striated flux patterns. The experimental identification of toroidal asymmetries in detached plasmas highlights the need for reliable 3D models for projecting the impact for ITER and beyond. Support from USDOE DE-AC05-00OR22725, DE-AC02-09CH11466, DE-FC02-99ER54512.

  10. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

    SciTech Connect

    Ko, Jae Hyung; Kim, Yang Hee; Jeong, Seong Hee; Lee, Song; Park, Si-Nae; Shim, In Kyong; Kim, Song Cheol

    2015-08-07

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.

  11. Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression

    PubMed Central

    Sawant, Sharada; Dongre, Harsh; Singh, Archana Kumari; Joshi, Shriya; Costea, Daniela Elena; Mahadik, Snehal; Ahire, Chetan; Makani, Vidhi; Dange, Prerana; Sharma, Shilpi; Chaukar, Devendra; Vaidya, Milind

    2016-01-01

    To study multistep tumorigenesis process, there is a need of in-vitro 3D model simulating in-vivo tissue. Present study aimed to reconstitute in-vitro tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted in-vitro models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced in-vitro. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in in-vitro grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis. PMID:27501241

  12. Recapitulating the Tumor Ecosystem Along the Metastatic Cascade Using 3D Culture Models

    PubMed Central

    Kim, Jiyun; Tanner, Kandice

    2015-01-01

    Advances in cancer research have shown that a tumor can be likened to a foreign species that disrupts delicately balanced ecological interactions, compromising the survival of normal tissue ecosystems. In efforts to mitigate tumor expansion and metastasis, experimental approaches from ecology are becoming more frequently and successfully applied by researchers from diverse disciplines to reverse engineer and re-engineer biological systems in order to normalize the tumor ecosystem. We present a review on the use of 3D biomimetic platforms to recapitulate biotic and abiotic components of the tumor ecosystem, in efforts to delineate the underlying mechanisms that drive evolution of tumor heterogeneity, tumor dissemination, and acquisition of drug resistance. PMID:26284194

  13. Ultrafast holographic technique for 3D in situ documentation of cultural heritage

    NASA Astrophysics Data System (ADS)

    Frey, Susanne; Bongartz, Jens; Giel, Dominik M.; Thelen, Andrea; Hering, Peter

    2003-10-01

    A novel 3d reconstruction method for medical application has been applied for the examination and documentation of a 2000-year-old bog body. An ultra-fast pulsed holographic camera has been modified to allow imaging of the bog body from different views. Full-scale daylight copies of the master holograms give a detailed impressive three-dimensional view of the mummy and can be exhibited instead of the object. In combination with a rapid prototyping model (built by the Rapid Prototyping group of the Stiftung caesar, Bonn, Germany) derived from computer tomography (CT) data our results are an ideal basis for a future facial reconstruction.

  14. Digital Inventory and Documentation of Korea's Important Cultural Properties Using 3D Laser Scanning

    NASA Astrophysics Data System (ADS)

    Dongseok, K.; Gyesoo, K.; Siro, K.; Eunhwa, K.

    2015-08-01

    As a country with 11 properties included on the World Heritage List and approximately 12,000 important cultural properties, Korea has been continuously carrying out the inventory and documentation of cultural properties to conserve and manage them since the 1960s. The inventory of cultural properties had been carried out by making and managing a register which recorded basic information mainly on state-designated cultural properties such as their size, quantity, and location. The documentation of cultural properties was also carried out by making measured drawings. However, the inventory and documentation done under the previous analog method had a limit to the information it could provide for the effective conservation and management of cultural properties. Moreover, in recent times important cultural properties have frequently been damaged by man-made and natural disasters such as arson, forest fires, and floods, so an alternative was required. Accordingly, Korea actively introduced digital techniques led by the government for the inventory and documentation of important cultural properties. In this process, the government established the concept of a digital set, built a more efficie nt integrated data management system, and created standardized guidelines to maximize the effectiveness of data acquisition, management, and utilization that greatly increased the level of digital inventory, documentation, and archiving.

  15. Comparison of the Expression of Hepatic Genes by Human Wharton’s Jelly Mesenchymal Stem Cells Cultured in 2D and 3D Collagen Culture Systems

    PubMed Central

    Khodabandeh, Zahra; Vojdani, Zahra; Talaei-Khozani, Tahereh; Jaberipour, Mansoureh; Hosseini, Ahmad; Bahmanpour, Soghra

    2016-01-01

    Background: Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) express liver-specific markers such as albumin, alpha-fetoprotein, cytokeratin-19, cytokeratin-18, and glucose-6-phosphatase. Therefore, they can be considered as a good source for cell replacement therapy for liver diseases. This study aimed to evaluate the effects of various culture systems on the hepatocyte-specific gene expression pattern of naïve HWJMSCs. Methods: HWJMSCs were characterized as MSCs by detecting the surface CD markers and capability to differentiate toward osteoblast and adipocyte. HWJMSCs were cultured in 2D collagen films and 3D collagen scaffolds for 21 days and were compared to control cultures. Real time RT-PCR was used to evaluate the expression of liver-specific genes. Results: The HWJMSCs which were grown on non-coated culture plates expressed cytokeratin-18 and -19, alpha-fetoprotein, albumin, glucose-6-phosphatase, and claudin. The expression of the hepatic nuclear factor 4 (HNF4) was very low. The cells showed a significant increase in caludin expression when they cultured in 3D collagen scaffolds compared to the conventional monolayer culture and 2D collagen scaffold. Conclusion: Various culture systems did not influence on hepatocyte specific marker expression by HWJMSCs, except for claudin. The expression of claudin showed that 3D collagen scaffold provided the extracellular matrix for induction of the cells to interconnect with each other. PMID:26722142

  16. Flexible simulation strategy for modeling 3D cultural objects based on multisource remotely sensed imagery

    NASA Astrophysics Data System (ADS)

    Guienko, Guennadi; Levin, Eugene

    2003-01-01

    New ideas and solutions never come alone. Although automated feature extraction is not sufficiently mature to move from the realm of scientific investigation into the category of production technology, a new goal has arisen: 3D simulation of real-world objects, extracted from images. This task, which evolved from feature extraction and is not an easy task itself, becomes even more complex, multi-leveled, and often uncertain and fuzzy when one exploits time-sequenced multi-source remotely sensed visual data. The basic components of the process are familiar image processing tasks: fusion of various types of imagery, automatic recognition of objects, removng those objects from the source images, and replacing them in the images with their realistic simulated "twin" object rendering. This paper discusses how to aggregate the most appropriate approach to each task into one technological process in order to develop a Manipulator for Visual Simulation of 3D objects (ManVIS) that is independent or imagery/format/media. The technology could be made general by combining a number of competent special purpose algorithms under appropriate contextual, geometric, spatial, and temporal constraints derived from a-priori knowledge. This could be achieved by planning the simulation in an Open Structure Simulation Strategy Manager (O3SM) a distinct component of ManVIS building the simulation strategy before beginning actual image manipulation.

  17. Recording, Visualization and Documentation of 3D Spatial Data for Monitoring Topography in Areas of Cultural Heritage

    NASA Astrophysics Data System (ADS)

    Maravelakis, Emmanouel; Konstantaras, Antonios; Axaridou, Anastasia; Chrysakis, Ioannis; Xinogalos, Michalis

    2014-05-01

    This research investigates the application of new system for 3D documentation of land degradation and its effect [1,2] on areas of cultural heritage via complete 3D data acquisition, 3D modeling and metadata recording using terrestrial laser scanners (TLS) [3,4,5]. As land degradation progresses through time it is important to be able to map and exactly replicate with great precision the entire 3D shape of the physical objects of interest, such as landslides, ground erosion, river boundaries, mad accumulation, etc. [1,2] TLS enables the extraction and recording of a very large number of points in space with great precision and without the need for any physical contact with the object of interest. Field specialists can then examine the produced models and comment on them both on the overall object of interest and on specific features of it by inserting annotations on certain parts of the model [6]. This process could be proven to be very cost effective as it can be repeated as often as necessary and produce a well catalogued documentation of the progress of land degradation at particular areas. The problem with repeating TLS models lies on the various types of hardware equipment and software systems that might be used for the extraction of point clouds, and the different people that might be called to analyze the findings. These often result in a large volume of interim and final products with little if no standardization, multiple different metadata and vague documentation [7], which makes metadata recordings [8] crucial both for one scientist to be able to follow upon the work of the other as well as being able to repeat the same work when deemed necessary. This makes the need for a repository tool proposed by the authors essential in order to record all work that is done in every TLS scanning, and makes the technology accessible to scientists of various different fields [9,10], eg. geologists, physicists, topographers, remote sensing engineers, archaeologists etc

  18. Nonlinear 3D projection printing of concave hydrogel microstructures for long-term multicellular spheroid and embryoid body culture.

    PubMed

    Hribar, K C; Finlay, D; Ma, X; Qu, X; Ondeck, M G; Chung, P H; Zanella, F; Engler, A J; Sheikh, F; Vuori, K; Chen, S C

    2015-06-01

    Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propagation. Here, we used a continuous 3D projection printing approach - with an important modification of nonlinear exposure - to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs. PMID:25900329

  19. Nonlinear 3D Projection Printing of Concave Hydrogel Microstructures for Long-Term Multicellular Spheroid and Embryoid Body Culture

    PubMed Central

    Hribar, K.C; Finlay, D.; Ma, X.; Qu, X.; Ondeck, M. G.; Chung, P. H.; Zanella, F.; Engler, A. J.; Sheikh, F.; Vuori, K.; Chen, S.

    2015-01-01

    Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propogation. Here, we used a continuous 3D projection printing approach – with an important modification of nonlinear exposure — to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs. PMID:25900329

  20. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    SciTech Connect

    Kim, Sun-Ah; Lee, Eun Kyung; Kuh, Hyo-Jeong

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  1. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

    PubMed Central

    Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

    2013-01-01

    Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells. PMID:24312827

  2. Culturing thick brain slices: An interstitial 3D microperfusion system for enhanced viability

    PubMed Central

    Rambani, Komal; Vukasinovic, Jelena; Glezer, Ari; Potter, Steve M.

    2009-01-01

    Brain slice preparations are well-established models for a wide spectrum of in vitro investigations in the neuroscience discipline. However, these investigations are limited to acute preparations or thin organotypic culture preparations due to the lack of a successful method that allows culturing of thick organotypic brain slices. Thick brain slice cultures suffer necrosis due to ischemia deep in the tissue resulting from a destroyed circulatory system and subsequent diffusion-limited supply of nutrients and oxygen. Although thin organotypic brain slice cultures can be successfully cultured using a well established roller tube method (a monolayer organotypic culture) (Gahwiler B H, 1981) or a membrane insert method (up to 1–4 cell layers, <150μm)(Stoppini L et al., 1991), these methods fail to support thick tissue preparations. A few perfusion methods (using submerged or interface/microfluidic chambers) have been reported to enhance the longevity (up to few hours) of acute slice preparations (up to 600μm thick) (Hass H L et al., 1979; Nicoll R A and Alger B E, 1981; Passeraub P A et al., 2003). Here, we report a unique interstitial microfluidic perfusion technique to culture thick (700μm) organotypic brain slices. The design of the custom-made micro-perfusion chamber facilitates laminar, interstitial perfusion of oxygenated nutrient medium throughout the tissue thickness with concomitant removal of depleted medium and catabolites. We examined the utility of this perfusion method to enhance the viability of the thick organotypic brain slice cultures after 2 days and 5 days in vitro (DIV). We investigated the range of amenable flow rates that enhance the viability of 700μm thick organotypic brain slices compared to the unperfused control cultures. Our perfusion method allows up to 84.6% viability (P<0.01) and up to 700μm thickness, even after 5 DIV. Our results also confirm that these cultures are functionally active and have their in vivo cytoarchitecture

  3. Does spatial variation in environmental conditions affect recruitment? A study using a 3-D model of Peruvian anchovy

    NASA Astrophysics Data System (ADS)

    Xu, Yi; Rose, Kenneth A.; Chai, Fei; Chavez, Francisco P.; Ayón, Patricia

    2015-11-01

    We used a 3-dimensional individual-based model (3-D IBM) of Peruvian anchovy to examine how spatial variation in environmental conditions affects larval and juvenile growth and survival, and recruitment. Temperature, velocity, and phytoplankton and zooplankton concentrations generated from a coupled hydrodynamic Nutrients-Phytoplankton-Zooplankton-Detritus (NPZD) model, mapped to a three dimensional rectangular grid, were used to simulate anchovy populations. The IBM simulated individuals as they progressed from eggs to recruitment at 10 cm. Eggs and yolk-sac larvae were followed hourly through the processes of development, mortality, and movement (advection), and larvae and juveniles were followed daily through the processes of growth, mortality, and movement (advection plus behavior). A bioenergetics model was used to grow larvae and juveniles. The NPZD model provided prey fields which influence both food consumption rate as well as behavior mediated movement with individuals going to grids cells having optimal growth conditions. We compared predicted recruitment for monthly cohorts for 1990 through 2004 between the full 3-D IBM and a point (0-D) model that used spatially-averaged environmental conditions. The 3-D and 0-D versions generated similar interannual patterns in monthly recruitment for 1991-2004, with the 3-D results yielding consistently higher survivorship. Both versions successfully captured the very poor recruitment during the 1997-1998 El Niño event. Higher recruitment in the 3-D simulations was due to higher survival during the larval stage resulting from individuals searching for more favorable temperatures that lead to faster growth rates. The strong effect of temperature was because both model versions provided saturating food conditions for larval and juvenile anchovies. We conclude with a discussion of how explicit treatment of spatial variation affected simulated recruitment, other examples of fisheries modeling analyses that have used a

  4. Web-based Visualization and Query of semantically segmented multiresolution 3D Models in the Field of Cultural Heritage

    NASA Astrophysics Data System (ADS)

    Auer, M.; Agugiaro, G.; Billen, N.; Loos, L.; Zipf, A.

    2014-05-01

    Many important Cultural Heritage sites have been studied over long periods of time by different means of technical equipment, methods and intentions by different researchers. This has led to huge amounts of heterogeneous "traditional" datasets and formats. The rising popularity of 3D models in the field of Cultural Heritage in recent years has brought additional data formats and makes it even more necessary to find solutions to manage, publish and study these data in an integrated way. The MayaArch3D project aims to realize such an integrative approach by establishing a web-based research platform bringing spatial and non-spatial databases together and providing visualization and analysis tools. Especially the 3D components of the platform use hierarchical segmentation concepts to structure the data and to perform queries on semantic entities. This paper presents a database schema to organize not only segmented models but also different Levels-of-Details and other representations of the same entity. It is further implemented in a spatial database which allows the storing of georeferenced 3D data. This enables organization and queries by semantic, geometric and spatial properties. As service for the delivery of the segmented models a standardization candidate of the OpenGeospatialConsortium (OGC), the Web3DService (W3DS) has been extended to cope with the new database schema and deliver a web friendly format for WebGL rendering. Finally a generic user interface is presented which uses the segments as navigation metaphor to browse and query the semantic segmentation levels and retrieve information from an external database of the German Archaeological Institute (DAI).

  5. Controllable Production of Transplantable Adult Human High-Passage Dermal Papilla Spheroids Using 3D Matrigel Culture

    PubMed Central

    Miao, Yong; Sun, Ya Bin; Liu, Bing Cheng; Jiang, Jin Dou

    2014-01-01

    We have succeeded in culturing human dermal papilla (DP) cell spheroids and developed a three-dimensional (3D) Matrigel (basement membrane matrix) culture technique that can enhance and restore DP cells unique characteristics in vitro. When 1×104 DP cells were cultured on the 96-well plates precoated with Matrigel for 5 days, both passage 2 and passage 8 DP cells formed spheroidal microtissues with a diameter of 150–250 μm in an aggregative and proliferative manner. We transferred and recultured these DP spheroids onto commercial plates. Cells within DP spheres could disaggregate and migrate out, which was similar to primary DP. Moreover, we examined the expression of several genes and proteins associated with hair follicle inductivity of DP cells, such as NCAM, Versican, and α-smooth muscle actin, and confirmed that their expression level was elevated in the spheres compared with the dissociated DP cells. To examine the hair-inducing ability of DP spheres, hair germinal matrix cells (HGMCs) and DP spheres were mixed and cultured on Matrigel. Unlike the dissociated DP cells and HGMCs cocultured in two dimensions, HGMCs can differentiate into hair-like fibers under the induction of the DP spheres made from the high-passage cells (passage 8) in vitro. We are the first to show that passage 3 human HGMCs differentiate into hair-like fibers in the presence of human DP spheroids. These results suggest that the 3D Matrigel culture technique is an ideal culture model for forming DP spheroids and that sphere formation partially models the intact DP, resulting in hair induction, even by high-passage DP cells. PMID:24528213

  6. High-Throughput Microfluidic Platform for 3D Cultures of Mesenchymal Stem Cells, Towards Engineering Developmental Processes.

    PubMed

    Occhetta, Paola; Centola, Matteo; Tonnarelli, Beatrice; Redaelli, Alberto; Martin, Ivan; Rasponi, Marco

    2015-01-01

    The development of in vitro models to screen the effect of different concentrations, combinations and temporal sequences of morpho-regulatory factors on stem/progenitor cells is crucial to investigate and possibly recapitulate developmental processes with adult cells. Here, we designed and validated a microfluidic platform to (i) allow cellular condensation, (ii) culture 3D micromasses of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) under continuous flow perfusion, and (ii) deliver defined concentrations of morphogens to specific culture units. Condensation of hBM-MSCs was obtained within 3 hours, generating micromasses in uniform sizes (56.2 ± 3.9 μm). As compared to traditional macromass pellet cultures, exposure to morphogens involved in the first phases of embryonic limb development (i.e. Wnt and FGF pathways) yielded more uniform cell response throughout the 3D structures of perfused micromasses (PMMs), and a 34-fold higher percentage of proliferating cells at day 7. The use of a logarithmic serial dilution generator allowed to identify an unexpected concentration of TGFβ3 (0.1 ng/ml) permissive to hBM-MSCs proliferation and inductive to chondrogenesis. This proof-of-principle study supports the described microfluidic system as a tool to investigate processes involved in mesenchymal progenitor cells differentiation, towards a 'developmental engineering' approach for skeletal tissue regeneration. PMID:25983217

  7. In vitro 3-D model based on extending time of culture for studying chronological epidermis aging.

    PubMed

    Dos Santos, Morgan; Metral, Elodie; Boher, Aurélie; Rousselle, Patricia; Thepot, Amélie; Damour, Odile

    2015-09-01

    Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the

  8. Three-dimensional Culture Conditions Lead to Decreased Radiation Induced Crytoxicity in Human Mammary Epithelial Cells

    SciTech Connect

    Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

    2010-05-01

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extra cellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D vs. 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ~4 fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures

  9. Responsive culture platform to examine the influence of microenvironmental geometry on cell function in 3D

    PubMed Central

    Kloxin, April M.; Lewis, Katherine J. R.; DeForest, Cole A.; Seedorf, Gregory; Tibbitt, Mark W.; Balasubramaniam, Vivek

    2012-01-01

    We describe the development of a well-based cell culture platform that enables experimenters to control the geometry and connectivity of cellular microenvironments spatiotemporally. The base material is a hydrogel comprised of photolabile and enzyme-labile crosslinks and pendant cell adhesion sequences, enabling spatially-specific, in situ patterning with light and cell-dictated microenvironment remodeling through enzyme secretion. Arrays of culture wells of varying shape and size were patterned into the hydrogel surface using photolithography, where well depth was correlated with irradiation dose. The geometry of these devices can be subsequently modified through sequential patterning, while simultaneously monitoring changes in cell geometry and connectivity. Towards establishing the utility of these devices for dynamic evaluation of the influence of physical cues on tissue morphogenesis, the effect of well shape on lung epithelial cell differentiation (i.e., primary mouse alveolar type II cells, ATII cells) was assessed. Shapes inspired by alveoli were degraded into hydrogel surfaces. ATII cells were seeded within the well-based arrays and encapsulated by the addition of a top hydrogel layer. Cell differentiation in response to these geometries was characterized over 7 days of culture with immunocytochemistry (surfactant protein C, ATII; T1α protein, alveolar type I (ATI) differentiated epithelial cells) and confocal image analysis. Individual cell clusters were further connected by eroding channels between wells during culture via controlled two-photon irradiation. Collectively, these studies demonstrate the development and utility of responsive hydrogel culture devices to study how a range of microenvironment geometries of evolving shape and connectivity might influence or direct cell function. PMID:23138879

  10. The Representation of Cultural Heritage from Traditional Drawing to 3d Survey: the Case Study of Casamary's Abbey

    NASA Astrophysics Data System (ADS)

    Canciani, M.; Saccone, M.

    2016-06-01

    In 3D survey the aspects most discussed in the scientific community are those related to the acquisition of data from integrated survey (laser scanner, photogrammetric, topographic and traditional direct), rather than those relating to the interpretation of the data. Yet in the methods of traditional representation, the data interpretation, such as that of the philological reconstruction, constitutes the most important aspect. It is therefore essential in modern systems of survey and representation, filter the information acquired. In the system, based on the integrated survey that we have adopted, the 3D object, characterized by a cloud of georeferenced points, defined but their color values, defines the core of the elaboration. It allows to carry out targeted analysis, using section planes as a tool of selection and filtering data, comparable with those of traditional drawings. In the case study of the Abbey of Casamari (Veroli), one of the most important Cistercian Settlement in Italy, the survey made for an Agreement with the Ministry of Cultural Heritage and Activities and Tourism (MiBACT) and University of RomaTre, within the project "Accessment of the sismic safety of the state museum", the reference 3D model, consisting of the superposition and geo-references data from various surveys, is the tool with which yo develop representative models comparable to traditional ones. It provides the necessary spatial environment for drawing up plans and sections with a definition such as to develop thematic analysis related to phases of construction, state of deterioration and structural features.

  11. Recording, Visualization and Documentation of 3D Spatial Data for Monitoring Topography in Areas of Cultural Heritage

    NASA Astrophysics Data System (ADS)

    Maravelakis, Emmanouel; Konstantaras, Antonios; Axaridou, Anastasia; Chrysakis, Ioannis; Xinogalos, Michalis

    2014-05-01

    This research investigates the application of new system for 3D documentation of land degradation and its effect [1,2] on areas of cultural heritage via complete 3D data acquisition, 3D modeling and metadata recording using terrestrial laser scanners (TLS) [3,4,5]. As land degradation progresses through time it is important to be able to map and exactly replicate with great precision the entire 3D shape of the physical objects of interest, such as landslides, ground erosion, river boundaries, mad accumulation, etc. [1,2] TLS enables the extraction and recording of a very large number of points in space with great precision and without the need for any physical contact with the object of interest. Field specialists can then examine the produced models and comment on them both on the overall object of interest and on specific features of it by inserting annotations on certain parts of the model [6]. This process could be proven to be very cost effective as it can be repeated as often as necessary and produce a well catalogued documentation of the progress of land degradation at particular areas. The problem with repeating TLS models lies on the various types of hardware equipment and software systems that might be used for the extraction of point clouds, and the different people that might be called to analyze the findings. These often result in a large volume of interim and final products with little if no standardization, multiple different metadata and vague documentation [7], which makes metadata recordings [8] crucial both for one scientist to be able to follow upon the work of the other as well as being able to repeat the same work when deemed necessary. This makes the need for a repository tool proposed by the authors essential in order to record all work that is done in every TLS scanning, and makes the technology accessible to scientists of various different fields [9,10], eg. geologists, physicists, topographers, remote sensing engineers, archaeologists etc

  12. Role of differential physical properties in emergent behavior of 3D cell co-cultures

    NASA Astrophysics Data System (ADS)

    Kolbman, Dan; Das, Moumita

    2015-03-01

    The biophysics of binary cell populations is of great interest in many biological processes, whether the formation of embryos or the initiation of tumors. During these processes, cells are surrounded by other cell types with different physical properties, often with important consequences. For example, recent experiments on a co-culture of breast cancer cells and healthy breast epithelial cells suggest that the mechanical mismatch between the two cell types may contribute to enhanced migration of the cancer cells. Here we explore how the differential physical properties of different cell types may influence cell-cell interaction, aggregation, and migration. To this end, we study a proof of concept model- a three-dimensional binary system of interacting, active, and deformable particles with different physical properties such as elastic stiffness, contractility, and particle-particle adhesion, using Langevin Dynamics simulations. Our results may provide insights into emergent behavior such as segregation and differential migration in cell co-cultures in three dimensions.

  13. Prognostic breast cancer signature identified from 3D culture model accurately predicts clinical outcome across independent datasets

    SciTech Connect

    Martin, Katherine J.; Patrick, Denis R.; Bissell, Mina J.; Fournier, Marcia V.

    2008-10-20

    One of the major tenets in breast cancer research is that early detection is vital for patient survival by increasing treatment options. To that end, we have previously used a novel unsupervised approach to identify a set of genes whose expression predicts prognosis of breast cancer patients. The predictive genes were selected in a well-defined three dimensional (3D) cell culture model of non-malignant human mammary epithelial cell morphogenesis as down-regulated during breast epithelial cell acinar formation and cell cycle arrest. Here we examine the ability of this gene signature (3D-signature) to predict prognosis in three independent breast cancer microarray datasets having 295, 286, and 118 samples, respectively. Our results show that the 3D-signature accurately predicts prognosis in three unrelated patient datasets. At 10 years, the probability of positive outcome was 52, 51, and 47 percent in the group with a poor-prognosis signature and 91, 75, and 71 percent in the group with a good-prognosis signature for the three datasets, respectively (Kaplan-Meier survival analysis, p<0.05). Hazard ratios for poor outcome were 5.5 (95% CI 3.0 to 12.2, p<0.0001), 2.4 (95% CI 1.6 to 3.6, p<0.0001) and 1.9 (95% CI 1.1 to 3.2, p = 0.016) and remained significant for the two larger datasets when corrected for estrogen receptor (ER) status. Hence the 3D-signature accurately predicts breast cancer outcome in both ER-positive and ER-negative tumors, though individual genes differed in their prognostic ability in the two subtypes. Genes that were prognostic in ER+ patients are AURKA, CEP55, RRM2, EPHA2, FGFBP1, and VRK1, while genes prognostic in ER patients include ACTB, FOXM1 and SERPINE2 (Kaplan-Meier p<0.05). Multivariable Cox regression analysis in the largest dataset showed that the 3D-signature was a strong independent factor in predicting breast cancer outcome. The 3D-signature accurately predicts breast cancer outcome across multiple datasets and holds prognostic

  14. Assessment of different 3D culture systems to study tumor phenotype and chemosensitivity in pancreatic ductal adenocarcinoma.

    PubMed

    Zeeberg, Katrine; Cardone, Rosa Angela; Greco, Maria Raffaella; Saccomano, Mara; Nøhr-Nielsen, Asbjørn; Alves, Frauke; Pedersen, Stine Falsig; Reshkin, Stephan Joel

    2016-07-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a very poor prognosis, due to the influence of the tumor stroma, which promotes tumor growth, early invasion and chemoradiation resistance. Efforts to develop models for identifying novel anticancer therapeutic compounds have been hampered by the limited ability of in vitro models to mimic these in vivo tumor-stroma interactions. This has led to the development of various three-dimensional (3D) culture platforms recapitulating the in vivo tumor-stroma crosstalk and designed to better understand basic cancer processes and screen drug action. However, a consensus for different experimental 3D platforms is still missing in PDAC. We compared four PDAC cell lines of different malignancy grown in 2D monolayers to three of the more commonly used 3D techniques (ultralow adhesion concave microwells, Matrigel inclusion and organotypic systems) and to tumors derived from their orthotopic implantation in mice. In these 3D platforms, we observed that cells grow with very different tumor morphologies and the organotypic setting most closely resembles the tumor cytoarchitecture obtained by orthotopically implanting the four cell lines in mice. We then analyzed the molecular and cellular responses of one of these cell lines to epidermal growth factor receptor (EGFR) stimulation with EGF and inhibition with erlotinib and found that only in the 3D platforms, and especially the organotypic, cells: i) responded to EGF by changing the expression of signalling components underlying cell-stroma crosstalk and tissue architecture, growth, invasion and drug resistance (E-cadherin, EGFR, ezrin, β1 integrin, NHERF1 and HIF-1α) similar to those reported in vivo; ii) had stimulated growth and increased erlotinib sensitivity in response to EGF, more faithfully mimicking their known in vivo behaviour. Altogether, these results, indicate the organotypic as the most relevant physiological 3D system to study the

  15. The myofibroblast markers α-SM actin and β-actin are differentially expressed in 2 and 3-D culture models of fibrotic and normal skin.

    PubMed

    Vozenin, M C; Lefaix, J L; Ridi, R; Biard, D S; Daburon, F; Martin, M

    1998-01-01

    To characterize the differences between fibrotic myofibroblasts and normal fibroblasts, we studied two differentiation markers: α-smooth muscle (SM) actin, a specific marker of myofibroblast differentiation, and β-actin, which is overexpressed in the fibrotic tissue. Experiments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fibrosis. Three culture models were used: cells in monolayers, equivalent dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Samples were studied using immunofluorescence and western-blotting. In monolayers cultures, both fibrosis and normal cells expressed α-SM actin. Furthermore, similar amounts of β-actin protein were found. In these conditions, the resulting alterations in the phenotypes of cells made comparison of cultured fibrotic and normal cells irrelevant. Under the two 3-D culture models, normal fibroblasts no longer expressed α-SM actin. They expressed β-actin at the basal level. Moreover, the fibrotic myofibroblasts in both 3-D models retained their differentiation features, expressing α-SM actin and overexpressing β-actin. We found that this normalization was mainly related to the genomic programmation acquired by the cells in the tissue. Cellular motility and microenvironment were also involved, whereas cellular proliferation was not a major factor. Consequently, both three-dimensional models allowed the study of radiation-induced fibrosis in vitro, provided good extrapolations to in vivo conditions and avoided certain of culture artefacts. PMID:22359004

  16. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons.

    PubMed

    Wardyn, Joanna D; Sanderson, Chris; Swan, Laura E; Stagi, Massimiliano

    2015-08-15

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology. PMID:25962333

  17. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons

    PubMed Central

    Wardyn, Joanna D.; Sanderson, Chris; Swan, Laura E.; Stagi, Massimiliano

    2015-01-01

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology. PMID:25962333

  18. [Novel methods for studies of testicular development and spermatogenesis: From 2D to 3D culture].

    PubMed

    Zhang, Lian-dong; Li, He-cheng; Zhang, Tong-dian; Wang, Zi-ming

    2016-03-01

    The two-dimensional model of cell culture is an important method in the study of testicular development and spermatogenesis but can not effectively mimic and regulate the testicular microenvironment and the whole process of spermatogenesis due to the lack of relevant cell factors and the disruption of a three-dimensional spatial structure. In the past 20 years, the development and optimization of the in vitro model such as testis organotypic culture and in vivo model such as testis transplantation achieved a transformation from two- to three-dimension. The maintenance and optimization of the testicular niche structure could mimic the testicular microenvironment and cell types including Leydig, Sertoli and germ cells, which showed similar biological behaviors to those in vivo. Besides, the cell suspension or tissue fragment floats in the gas-liquid interface so that the development of somatic and germ cells is well maintained in vitro whilst the feedback linkage between grafted testis tissue and hypothalamus-pituitary of the host rebuilt in the in vitro model provides an endocrinological basis for spermatogenesis, which serves as an effective methodology to better understand the organogenesis and development of the testis as well as testicular function regulation, advancing the concept of treatment of male infertility. Al- though each of the methods may have its limitations, the progress in the processing, freezing, thawing, and transplantation of cells and tissues will surely promote their clinical application and present their value in translational medicine. PMID:27172668

  19. Type conversion of secretomes in a 3D TAM2 and HCC cell co-culture system and functional importance of CXCL2 in HCC

    PubMed Central

    Lu, Yu; Li, Shan; Ma, Liping; Li, Yan; Zhang, Xiaolian; Peng, Qiliu; Mo, Cuiju; Huang, Li; Qin, Xue; Liu, Yinkun

    2016-01-01

    Macrophages play important roles in the tumor microenvironment, driving cancer progression and metastasis, particularly in hepatocellular carcinoma (HCC). However, few studies have assessed the exact secretome composition in HCC. In the present study, the impact of different phenotype of macrophages on HCC cells was investigated. Alternatively activated macrophages (M2) were found to significantly increase the proliferation, migration, and invasion abilities of SMMC7721 cells (all P < 0.05). M2 were then co-cultured with SMMC7721 cells to reconstruct the tumor microenvironment. Conditioned medium from 3D single cultures of M2, SMMC7721 cells, and their co-culture system were analyzed using quantitative proteomics via iTRAQ labeling combined with mass spectrometric analysis. Secretome analysis revealed a total of 159 differential secreted proteins in the co-culture system compared to the single culture systems, with 63 being up-regulated (>1.3-fold) and 96 down-regulated (<0.7-fold). CXCL2 was confirmed to have higher expression in the co-culture system and HCC tissues, and was selected for further investigation. Functional effects data suggested that recombinant human CXCL2 significantly enhanced the migration, invasion ability of SMMC7721 cells, and weakened adhesion ability. While CXCL2 neutralization and CXCR2 blockage significantly inhibited the effects of CXCL2 on SMMC7721 cells, indicating that CXCL2 may play pivotal role in HCC metastasis. PMID:27117207

  20. Testing the Low-Cost Rpas Potential in 3d Cultural Heritage Reconstruction

    NASA Astrophysics Data System (ADS)

    Bolognesi, M.; Furini, A.; Russo, V.; Pellegrinelli, A.; Russo, P.

    2015-02-01

    In order to analyze the potential as well as the limitations of low-cost RPAS photogrammetric systems for architectural cultural heritage reconstruction, some tests were performed by a small RPAS equipped with an ultralight camera. The tests were carried out in a site of remarkable historical interest. A great amount of images were taken with camera's optical axis in vertical and oblique position. Images were processed by the commercial software PhotoScan of Agisoft and numerous models were realized, each of them was compared with an accurate TLS model used as a reference. The test, despite some problems found, has provided good results in terms of accuracy (average error <2cm) and reliability.

  1. Impact assessment of repeated exposure of organotypic 3D bronchial and nasal tissue culture models to whole cigarette smoke.

    PubMed

    Kuehn, Diana; Majeed, Shoaib; Guedj, Emmanuel; Dulize, Remi; Baumer, Karine; Iskandar, Anita; Boue, Stephanie; Martin, Florian; Kostadinova, Radina; Mathis, Carole; Ivanov, Nikolai V; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C

    2015-01-01

    Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers. PMID:25741927

  2. Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

    PubMed Central

    Kuehn, Diana; Majeed, Shoaib; Guedj, Emmanuel; Dulize, Remi; Baumer, Karine; Iskandar, Anita; Boue, Stephanie; Martin, Florian; Kostadinova, Radina; Mathis, Carole; Ivanov, Nikolai V.; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C.

    2015-01-01

    Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers. PMID:25741927

  3. Change of Re dependency of single bubble 3D motion by surface slip condition in surfactant solution

    NASA Astrophysics Data System (ADS)

    Tagawa, Yoshiyuki; Funakubo, Ami; Takagi, Shu; Matsumoto, Yoichiro

    2009-11-01

    Path instability of single bubble in water is sensitive to surfactant. One of the key effects of surfactant is to decrease bubble rising velocity (i.e. increase drag) and change bubble slip condition from free-slip to no-slip. This phenomenon is described as Marangoni effect. However, the surfactant effect to path instability is not fully investigated. In this research, we measured bubble 3D trajectories and velocity in dilute surfactant solution to reveal the relation between 3D motion mode and slip condition. Experimental parameters are types of surfactants, concentrations and bubble sizes. Bubble motions categorized as straight, spiral or zigzag are plotted on two-dimensional field of bubble Reynolds number Re and normalized drag coefficient CD^* which is strongly related to surface slip condition. Range of Re is from 200 to 1000 and CD^* is from 0 to 1. Our results show that when CD^* equals 0 or 1 (free-slip condition or no-slip condition, respectively), bubble motion mode is changed by Re. However when CD^* is 0.5, bubble motion is always spiral. It means that Re dependency of bubble motions is strongly affected by slip condition. We will discuss its mechanism in detail in our presentation.

  4. Multiplex profiling of cellular invasion in 3D cell culture models.

    PubMed

    Burgstaller, Gerald; Oehrle, Bettina; Koch, Ina; Lindner, Michael; Eickelberg, Oliver

    2013-01-01

    To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states. PMID:23671660

  5. How linguistic and cultural forces shape conceptions of time: English and Mandarin time in 3D.

    PubMed

    Fuhrman, Orly; McCormick, Kelly; Chen, Eva; Jiang, Heidi; Shu, Dingfang; Mao, Shuaimei; Boroditsky, Lera

    2011-01-01

    In this paper we examine how English and Mandarin speakers think about time, and we test how the patterns of thinking in the two groups relate to patterns in linguistic and cultural experience. In Mandarin, vertical spatial metaphors are used more frequently to talk about time than they are in English; English relies primarily on horizontal terms. We present results from two tasks comparing English and Mandarin speakers' temporal reasoning. The tasks measure how people spatialize time in three-dimensional space, including the sagittal (front/back), transverse (left/right), and vertical (up/down) axes. Results of Experiment 1 show that people automatically create spatial representations in the course of temporal reasoning, and these implicit spatializations differ in accordance with patterns in language, even in a non-linguistic task. Both groups showed evidence of a left-to-right representation of time, in accordance with writing direction, but only Mandarin speakers showed a vertical top-to-bottom pattern for time (congruent with vertical spatiotemporal metaphors in Mandarin). Results of Experiment 2 confirm and extend these findings, showing that bilinguals' representations of time depend on both long-term and proximal aspects of language experience. Participants who were more proficient in Mandarin were more likely to arrange time vertically (an effect of previous language experience). Further, bilinguals were more likely to arrange time vertically when they were tested in Mandarin than when they were tested in English (an effect of immediate linguistic context). PMID:21884222

  6. Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models

    PubMed Central

    Burgstaller, Gerald; Oehrle, Bettina; Koch, Ina; Lindner, Michael; Eickelberg, Oliver

    2013-01-01

    To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states. PMID:23671660

  7. Establishment of 3D organotypic cultures using human neonatal epidermal cells.

    PubMed

    Gangatirkar, Pradnya; Paquet-Fifield, Sophie; Li, Amy; Rossi, Ralph; Kaur, Pritinder

    2007-01-01

    This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air-liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 mum) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes. PMID:17401352

  8. Assessment of the developmental toxicity of nanoparticles in an ex vivo 3D model, the murine limb bud culture system.

    PubMed

    Bigaeva, Emilia; Paradis, France-Hélène; Moquin, Alexandre; Hales, Barbara F; Maysinger, Dusica

    2015-01-01

    The rapid growth of nanotechnological products for biomedical applications has exacerbated the need for suitable biological tests to evaluate the potential toxic effects of nanomaterials. The possible consequences of exposure during embryo and fetal development are of particular concern. The limb bud culture is an ex vivo 3D model in which growth, cell differentiation, and tissue organization occur and both molecular and functional endpoints can be quantitatively assessed. We employed this model to assess biochemical and morphological changes induced during organogenesis by two classes of nanostructured materials: quantum dot nanocrystals and organic polyglycerol sulfate dendrimers (dPGS). We show that quantum dots carrying mercaptopropionic acid (QD-MPA) on the surface, commonly used in biological studies, inhibit the development of limb buds from CD1 wildtype and Col2a1; Col10a1; Col1a1 triple transgenic fluorescent reporter mice, as revealed by changes in several morphological and biochemical markers. QD-MPA interfere with chondrogenesis and osteogenesis and disrupt the expression of COL10A1 and COL1A1, key markers of differentiation. In contrast, equivalent (3-100 nM) concentrations of dPGS do not adversely affect limb development. Neither QD-MPA nor dPGS-Cy5 alters the expression of several markers of cell proliferation or apoptosis. Collectively, these results suggest that murine limb buds in culture constitute a convenient, inexpensive and reliable developmental model for the assessment of the nanotoxicological effects of nanocrystals and polymers. In these 3D cultures, any effect that is observed can be directly ascribed to the nanostructures per se or a degradation component released from the complex nanostructure. PMID:25387253

  9. The role of 3D microenvironmental organization in MCF-7 epithelial–mesenchymal transition after 7 culture days

    SciTech Connect

    Foroni, Laura; Vasuri, Francesco; Valente, Sabrina; Gualandi, Chiara; Focarete, Maria Letizia; Caprara, Giacomo; Scandola, Mariastella; D'Errico-Grigioni, Antonia; Pasquinelli, Gianandrea

    2013-06-10

    We present a multi-technique study on in vitro epithelial–mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(L-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, β-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, β-catenin and vimentin) and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and β-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-β2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold. -- Highlights: • After 7 culture days an aligned-PLA scaffold induces a spindle shape to MCF-7 cells. • Despite these changes, the aligned MCF-7 cells keep an epithelial phenotype. • The extracellular environment alone influences the E-cadherin/β-catenin axis. • The extracellular environment can promote the epithelial–mesenchymal transition.

  10. 3-D simulations to investigate initial condition effects on the growth of Rayleigh-Taylor mixing

    SciTech Connect

    Andrews, Malcolm J

    2008-01-01

    The effect of initial conditions on the growth rate of turbulent Rayleigh-Taylor (RT) mixing has been studied using carefully formulated numerical simulations. An integrated large-eddy simulation (ILES) that uses a finite-volume technique was employed to solve the three-dimensional incompressible Euler equations with numerical dissipation. The initial conditions were chosen to test the dependence of the RT growth parameters ({alpha}{sub b}, {alpha}{sub s}) on variations in (a) the spectral bandwidth, (b) the spectral shape, and (c) discrete banded spectra. Our findings support the notion that the overall growth of the RT mixing is strongly dependent on initial conditions. Variation in spectral shapes and bandwidths are found to have a complex effect of the late time development of the RT mixing layer, and raise the question of whether we can design RT transition and turbulence based on our choice of initial conditions. In addition, our results provide a useful database for the initialization and development of closures describing RT transition and turbulence.

  11. ISIM3D: AN ANSI-C THREE-DIMENSIONAL MULTIPLE INDICATOR CONDITIONAL SIMULATION PROGRAM

    EPA Science Inventory

    The indicator conditional simulation technique provides stochastic simulations of a variable that (i) honor the initial data and (ii) can feature a richer family of spatial structures not limited by Gaussianity. he data are encoded into a series of indicators which then are used ...

  12. Development of nanocellulose scaffolds with tunable structures to support 3D cell culture.

    PubMed

    Liu, Jun; Cheng, Fang; Grénman, Henrik; Spoljaric, Steven; Seppälä, Jukka; E Eriksson, John; Willför, Stefan; Xu, Chunlin

    2016-09-01

    Swollen three-dimensional nanocellulose films and their resultant aerogels were prepared as scaffolds towards tissue engineering application. The nanocellulose hydrogels with various swelling degree (up to 500 times) and the resultant aerogels with desired porosity (porosity up to 99.7% and specific surface area up to 308m(2)/g) were prepared by tuning the nanocellulose charge density, the swelling media conditions, and the material processing approach. Representative cell-based assays were applied to assess the material biocompatibility and efficacy of the human extracellular matrix (ECM)-mimicking nanocellulose scaffolds. The effects of charge density and porosity of the scaffolds on the biological tests were investigated for the first time. The results reveal that the nanocellulose scaffolds could promote the survival and proliferation of tumor cells, and enhance the transfection of exogenous DNA into the cells. These results suggest the usefulness of the nanocellulose-based matrices in supporting crucial cellular processes during cell growth and proliferation. PMID:27185139

  13. See-Through Imaging of Laser-Scanned 3d Cultural Heritage Objects Based on Stochastic Rendering of Large-Scale Point Clouds

    NASA Astrophysics Data System (ADS)

    Tanaka, S.; Hasegawa, K.; Okamoto, N.; Umegaki, R.; Wang, S.; Uemura, M.; Okamoto, A.; Koyamada, K.

    2016-06-01

    We propose a method for the precise 3D see-through imaging, or transparent visualization, of the large-scale and complex point clouds acquired via the laser scanning of 3D cultural heritage objects. Our method is based on a stochastic algorithm and directly uses the 3D points, which are acquired using a laser scanner, as the rendering primitives. This method achieves the correct depth feel without requiring depth sorting of the rendering primitives along the line of sight. Eliminating this need allows us to avoid long computation times when creating natural and precise 3D see-through views of laser-scanned cultural heritage objects. The opacity of each laser-scanned object is also flexibly controllable. For a laser-scanned point cloud consisting of more than 107 or 108 3D points, the pre-processing requires only a few minutes, and the rendering can be executed at interactive frame rates. Our method enables the creation of cumulative 3D see-through images of time-series laser-scanned data. It also offers the possibility of fused visualization for observing a laser-scanned object behind a transparent high-quality photographic image placed in the 3D scene. We demonstrate the effectiveness of our method by applying it to festival floats of high cultural value. These festival floats have complex outer and inner 3D structures and are suitable for see-through imaging.

  14. Mini-pillar array for hydrogel-supported 3D culture and high-content histologic analysis of human tumor spheroids.

    PubMed

    Kang, Jihoon; Lee, Dong Woo; Hwang, Hyun Ju; Yeon, Sang-Eun; Lee, Moo-Yeal; Kuh, Hyo-Jeong

    2016-06-21

    Three-dimensional (3D) cancer cell culture models mimic the complex 3D organization and microenvironment of human solid tumor tissue and are thus considered as highly predictive models representing avascular tumor regions. Confocal laser scanning microscopy is useful for monitoring drug penetration and therapeutic responses in 3D tumor models; however, photonic attenuation at increasing imaging depths and limited penetration of common fluorescence tracers are significant technical challenges to imaging. Immunohistological staining would be a good alternative, but the preparation of tissue sections from rather fragile spheroids through fixing and embedding procedures is challenging. Here we introduce a novel 3 × 3 mini-pillar array chip that can be utilized for 3D cell culturing and sectioning for high-content histologic analysis. The mini-pillar array chip facilitated the generation of 3D spheroids of human cancer cells within hydrogels such as alginate, collagen, and Matrigel. As expected, visualization of the 3D distribution of calcein AM and doxorubicin by optical sectioning was limited by photonic attenuation and dye penetration. The integrity of the 3D microtissue section was confirmed by immunostaining on paraffin sections and cryo-sections. The applicability of the mini-pillar array for drug activity evaluation was tested by measuring viability changes in spheroids exposed to anti-cancer agents, 5-fluorouracil and tirapazamine. Thus, our novel mini-pillar array platform can potentially promote high-content histologic analysis of 3D cultures and can be further optimized for field-specific needs. PMID:27194205

  15. Transition to Turbulence and Effect of Initial Conditions on 3D Compressible Mixing in Planar Blast-wave-driven Systems

    SciTech Connect

    Miles, A R; Edwards, M J; Greenough, J A

    2004-11-08

    Perturbations on an interface driven by a strong blast wave grow in time due to a combination of Rayleigh-Taylor, Richtmyer-Meshkov, and decompression effects. In this paper, results from three-dimensional numerical simulations of such a system under drive conditions to be attainable on the National Ignition Facility [E. M. Campbell, Laser Part. Beams, 9(2), 209 (1991)] are presented. Using the multi-physics, adaptive mesh refinement, higher order Godunov Eulerian hydrocode, Raptor [L. H. Howell and J.A. Greenough, J. Comp. Phys. 184, 53 (2003)], the late nonlinear instability evolution, including transition to turbulence, is considered for various multimode perturbation spectra. The 3D post-transition state differs from the 2D result, but the process of transition proceeds similarly in both 2D and 3D. The turbulent mixing transition results in a reduction in the growth rate of the mixing layer relative to its pre-transition value and, in the case of the bubble front, relative to the 2D result. The post-transition spike front velocity is approximately the same in 2D and 3D. Implications for hydrodynamic mixing in core-collapse supernova are discussed.

  16. 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. PMID:26608295

  17. A 3D ex vivo mandible slice system for longitudinal culturing of transplanted dental pulp progenitor cells

    PubMed Central

    Colombo, John S.; Young, Fraser I.; Waddington, Rachel J.; Errington, Rachel J.; Sloan, Alastair J.

    2015-01-01

    Abstract Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell‐lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the β‐actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP‐DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP‐DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell‐micro‐community, phenotypically modified in the tooth (the “biology”); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high‐content imaging (the biology in a “multiplex” format). © 2015 The Authors. Published by Wiley Periodicals, Inc. PMID:25963448

  18. Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system.

    PubMed

    Simon, Karen A; Mosadegh, Bobak; Minn, Kyaw Thu; Lockett, Matthew R; Mohammady, Marym R; Boucher, Diane M; Hall, Amy B; Hillier, Shawn M; Udagawa, Taturo; Eustace, Brenda K; Whitesides, George M

    2016-07-01

    This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation. PMID:27116031

  19. A 3D ex vivo mandible slice system for longitudinal culturing of transplanted dental pulp progenitor cells.

    PubMed

    Colombo, John S; Howard-Jones, Rachel A; Young, Fraser I; Waddington, Rachel J; Errington, Rachel J; Sloan, Alastair J

    2015-10-01

    Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell-lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the β-actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP-DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP-DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell-micro-community, phenotypically modified in the tooth (the "biology"); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high-content imaging (the biology in a "multiplex" format). PMID:25963448

  20. Electrosensitization assists cell ablation by nanosecond pulsed electric field in 3D cultures

    PubMed Central

    Muratori, Claudia; Pakhomov, Andrei G.; Xiao, Shu; Pakhomova, Olga N.

    2016-01-01

    Previous studies reported a delayed increase of sensitivity to electroporation (termed “electrosensitization”) in mammalian cells that had been subjected to electroporation. Electrosensitization facilitated membrane permeabilization and reduced survival in cell suspensions when the electric pulse treatments were split in fractions. The present study was aimed to visualize the effect of sensitization and establish its utility for cell ablation. We used KLN 205 squamous carcinoma cells embedded in an agarose gel and cell spheroids in Matrigel. A local ablation was created by a train of 200 to 600 of 300-ns pulses (50 Hz, 300–600 V) delivered by a two-needle probe with 1-mm inter-electrode distance. In order to facilitate ablation by engaging electrosensitization, the train was split in two identical fractions applied with a 2- to 480-s interval. At 400–600 V (2.9–4.3 kV/cm), the split-dose treatments increased the ablation volume and cell death up to 2–3-fold compared to single-train treatments. Under the conditions tested, the maximum enhancement of ablation was achieved when two fractions were separated by 100 s. The results suggest that engaging electrosensitization may assist in vivo cancer ablation by reducing the voltage or number of pulses required, or by enabling larger inter-electrode distances without losing the ablation efficiency. PMID:26987779

  1. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.

    PubMed

    Broutier, Laura; Andersson-Rolf, Amanda; Hindley, Christopher J; Boj, Sylvia F; Clevers, Hans; Koo, Bon-Kyoung; Huch, Meritxell

    2016-09-01

    Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol. PMID:27560176

  2. Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing.

    PubMed

    Imani, Rana; Hojjati Emami, Shahriar; Fakhrzadeh, Hossein; Baheiraei, Nafiseh; Sharifi, Ali M

    2012-04-01

    The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration. Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology, particularly in organ printing. Two simple methods, hanging drop (HD) and conical tube (CT) were utilized to prepare cell aggregates. The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared. The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated. In both methods, the optimum average size of the aggregates was less than 500 microm. CT aggregates were smaller than HD aggregates. 5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface. The proliferative ability reduced when the initial cell density was increased. Comparing these methods, we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability, spreading, and proliferation. In conclusion, smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique. PMID:23173303

  3. Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.

    PubMed

    Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei; Nakamura, Jutaro; Brooks, Matthew; Kaya, Koray Dogan; Chaitankar, Vijender; Michael, Sam; Tawa, Gregory; Zou, Jizhong; Rao, Mahendra; Zheng, Wei; Cogliati, Tiziana; Swaroop, Anand

    2016-04-01

    We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration. PMID:27116668

  4. Effect of Factors Secreted by the Placenta on Phenotype of THP-1 Cells Cultured on a 3D Scaffold.

    PubMed

    Lvova, T Yu; Stepanova, O I; Viazmina, L P; Okorokova, L S; Belyakova, K L; Belikova, M E; Selkov, S A; Sokolov, D I

    2016-05-01

    We studied the effects of secretory products of the placenta obtained from women with normal pregnancy and preeclampsia on the expression of surface markers by THP-1 cells cultured on a 3D Matrigel scaffold. Secretory products of third trimester placentas obtained from women with normal pregnancy reduced the relative number of THP-1 cells expressing CD54 and CD14 molecules and expression of CD14 and CD95 molecules by THP-1 cells in comparison with the effect of secretory products first trimester placentas. In parallel, the intensity of CD49d expression by THP-1 cells increased in the presence of secretory products of third trimester placentas in comparison with the first trimester. No differences in the expression of the studied molecules by THP-1 cells under the effect of placentas from women with physiological pregnancy and patients with preeclampsia were found. PMID:27259498

  5. Composite Scaffolds Containing Silk Fibroin, Gelatin, and Hydroxyapatite for Bone Tissue Regeneration and 3D Cell Culturing

    PubMed Central

    Moisenovich, M. M.; Arkhipova, A. Yu.; Orlova, A. A.; Drutskaya, M. S; Volkova, S. V.; Zacharov, S. E.; Agapov, I. I.; Kirpichnikov, M. P.

    2014-01-01

    Three-dimensional (3D) silk fibroin scaffolds were modified with one of the major bone tissue derivatives (nano-hydroxyapatite) and/or a collagen derivative (gelatin). Adhesion and proliferation of mouse embryonic fibroblasts (MEF) within the scaffold were increased after modification with either nano-hydroxyapatite or gelatin. However, a significant increase in MEF adhesion and proliferation was observed when both additives were introduced into the scaffold. Such modified composite scaffolds provide a new and better platform to study wound healing, bone and other tissue regeneration, as well as artificial organ bioengineering. This system can further be applied to establish experimental models to study cell-substrate interactions, cell migration and other complex processes, which may be difficult to address using the conventional two-dimensional culture systems. PMID:24772332

  6. Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state.

    PubMed

    Arranja, Alexandra; Denkova, Antonia G; Morawska, Karolina; Waton, Gilles; van Vlierberghe, Sandra; Dubruel, Peter; Schosseler, François; Mendes, Eduardo

    2016-02-28

    This work reveals how the physicochemical properties of Pluronic block copolymers influence significantly their interactions with cancer cells, whether in monolayer or spheroid cultures, and how different clinical applications can be foreseen. Two-dimensional (2D) and three-dimensional (3D) cell culture models were used to investigate the interactions of Pluronic carriers with different PEO block length and aggregation state (unimers versus cross-linked micelles) in HeLa and U87 cancer cells. Stabilized micelles of Pluronic P94 or F127 were obtained by polymerization of a crosslinking agent in the micelles hydrophobic core. Nanocarriers were functionalized with a fluorescent probe for visualization, and with a chelator for radiolabeling with Indium-111 and gamma-quantification. The 2D cell models revealed that the internalization pathways and ultimate cellular localization of the Pluronic nanocarriers depended largely on both the PEO block size and aggregation state of the copolymers. The smaller P94 unimers with an average radius of 2.1nm and the shortest PEO block mass (1100gmol(-1)) displayed the highest cellular uptake and retention. 3D tumor spheroids were used to assess the penetration capacity and toxicity potential of the nanocarriers. Results showed that cross-linked F127 micelles were more efficiently delivered across the tumor spheroids, and the penetration depth depends mostly on the transcellular transport of the carriers. The Pluronic P94-based carriers with the shortest PEO block length induced spheroid toxicity, which was significantly influenced by the spheroid cellular type. PMID:26792572

  7. 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression.

    PubMed

    Pinto, Sheena; Stark, Hans-Jürgen; Martin, Iris; Boukamp, Petra; Kyewski, Bruno

    2015-01-01

    Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance. Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80(lo), Aire-negative into CD80(hi), Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80(hi) mTECs. PMID:26275017

  8. The methodology of documenting cultural heritage sites using photogrammetry, UAV, and 3D printing techniques: the case study of Asinou Church in Cyprus

    NASA Astrophysics Data System (ADS)

    Themistocleous, K.; Ioannides, M.; Agapiou, A.; Hadjimitsis, D. G.

    2015-06-01

    As the affordability, reliability and ease-of-use of Unmanned Aerial Vehicles (UAV) advances, the use of aerial surveying for cultural heritage purposes becomes a popular choice, yielding an unprecedented volume of high-resolution, geo-tagged image-sets of historical sites from above. As well, recent developments in photogrammetry technology provide a simple and cost-effective method of generating relatively accurate 3D models from 2D images. These techniques provide a set of new tools for archaeologists and cultural heritage experts to capture, store, process, share, visualise and annotate 3D models in the field. This paper focuses on the methodology used to document the cultural heritage site of Asinou Church in Cyprus using various state of the art techniques, such as UAV, photogrammetry and 3D printing. Hundreds of images of the Asinou Church were taken by a UAV with an attached high resolution, low cost camera. These photographic images were then used to create a digital 3D model and a 3D printer was used to create a physical model of the church. Such a methodology provides archaeologists and cultural heritage experts a simple and cost-effective method of generating relatively accurate 3D models from 2D images of cultural heritage sites.

  9. Automatic Generation of Boundary Conditions Using Demons Nonrigid Image Registration for Use in 3-D Modality-Independent Elastography

    PubMed Central

    Ou, Jao J.; Ong, Rowena E.; Miga, Michael I.

    2013-01-01

    Modality-independent elastography (MIE) is a method of elastography that reconstructs the elastic properties of tissue using images acquired under different loading conditions and a biomechanical model. Boundary conditions are a critical input to the algorithm and are often determined by time-consuming point correspondence methods requiring manual user input. This study presents a novel method of automatically generating boundary conditions by nonrigidly registering two image sets with a demons diffusion-based registration algorithm. The use of this method was successfully performed in silico using magnetic resonance and X-ray-computed tomography image data with known boundary conditions. These preliminary results produced boundary conditions with an accuracy of up to 80% compared to the known conditions. Demons-based boundary conditions were utilized within a 3-D MIE reconstruction to determine an elasticity contrast ratio between tumor and normal tissue. Two phantom experiments were then conducted to further test the accuracy of the demons boundary conditions and the MIE reconstruction arising from the use of these conditions. Preliminary results show a reasonable characterization of the material properties on this first attempt and a significant improvement in the automation level and viability of the method. PMID:21690002

  10. Coupling curvature-dependent and shear stress-stimulated neotissue growth in dynamic bioreactor cultures: a 3D computational model of a complete scaffold.

    PubMed

    Guyot, Y; Papantoniou, I; Luyten, F P; Geris, L

    2016-02-01

    The main challenge in tissue engineering consists in understanding and controlling the growth process of in vitro cultured neotissues toward obtaining functional tissues. Computational models can provide crucial information on appropriate bioreactor and scaffold design but also on the bioprocess environment and culture conditions. In this study, the development of a 3D model using the level set method to capture the growth of a microporous neotissue domain in a dynamic culture environment (perfusion bioreactor) was pursued. In our model, neotissue growth velocity was influenced by scaffold geometry as well as by flow- induced shear stresses. The neotissue was modeled as a homogenous porous medium with a given permeability, and the Brinkman equation was used to calculate the flow profile in both neotissue and void space. Neotissue growth was modeled until the scaffold void volume was filled, thus capturing already established experimental observations, in particular the differences between scaffold filling under different flow regimes. This tool is envisaged as a scaffold shape and bioprocess optimization tool with predictive capacities. It will allow controlling fluid flow during long-term culture, whereby neotissue growth alters flow patterns, in order to provide shear stress profiles and magnitudes across the whole scaffold volume influencing, in turn, the neotissue growth. PMID:26758425

  11. Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins

    PubMed Central

    Edmondson, Rasheena; Adcock, Audrey F.; Yang, Liju

    2016-01-01

    This study investigated the effects of matrix on the behaviors of 3D-cultured cells of two prostate cancer cell lines, LNCaP and DU145. Two biologically-derived matrices, Matrigel and Cultrex BME, and one synthetic matrix, the Alvetex scaffold, were used to culture the cells. The cell proliferation rate, cellular response to anti-cancer drugs, and expression levels of proteins associated with drug sensitivity/resistance were examined and compared amongst the 3D-cultured cells on the three matrices and 2D-cultured cells. The cellular responses upon treatment with two common anti-cancer drugs, Docetaxel and Rapamycin, were examined. The expressions of epidermal growth factor receptor (EGFR) and β-III tubulin in DU145 cells and p53 in LNCaP cells were examined. The results showed that the proliferation rates of cells cultured on the three matrices varied, especially between the synthetic matrix and the biologically-derived matrices. The drug responses and the expressions of drug sensitivity-associated proteins differed between cells on various matrices as well. Among the 3D cultures on the three matrices, increased expression of β-III tubulin in DU145 cells was correlated with increased resistance to Docetaxel, and decreased expression of EGFR in DU145 cells was correlated with increased sensitivity to Rapamycin. Increased expression of a p53 dimer in 3D-cultured LNCaP cells was correlated with increased resistance to Docetaxel. Collectively, the results showed that the matrix of 3D cell culture models strongly influences cellular behaviors, which highlights the imperative need to achieve standardization of 3D cell culture technology in order to be used in drug screening and cell biology studies. PMID:27352049

  12. 75 FR 13238 - Special Conditions: McCauley Propeller Systems, Model Propeller 3D15C1401/C80MWX-X

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-19

    ...-SC, for McCauley Propeller Systems for model propeller 3D15C1401/C80MWX-X (71 FR 43674). On November... Propeller 3D15C1401/C80MWX-X AGENCY: Federal Aviation Administration (FAA), DOT. ACTION: Notice of proposed... proposed special conditions for McCauley Propeller Systems for model propeller 3D15C1401/C80MWX-X. We...

  13. Formulation and stability evaluation of 3D alginate beads potentially useful for cumulus-oocyte complexes culture.

    PubMed

    Dorati, Rossella; Genta, Ida; Ferrari, Michela; Vigone, Giulia; Merico, Valeria; Garagna, Silvia; Zuccotti, Maurizio; Conti, Bice

    2016-01-01

    Ovarian follicle encapsulation in synthetic or natural matrixes based on biopolymers is potentially a promising approach to in vitro maturation (IVM) process, since it maintains follicle 3D organisation by preventing its flattening and consequent disruption of gap junctions, preserving the functional relationship between oocyte and companion follicle cells. The aim of the work was to optimise physico-chemical parameters of alginate microcapsules for perspective IVM under 3D environments. On this purpose alginate and cross-linking agent concentrations were investigated. Alginate concentration between 0.75% and 0.125% w/w and Mg(2+), Ba(2+), Ca(2+ )at concentration between 100 and 20 mM were tested. Follicle encapsulation was obtained by on purpose modified diffusion setting gelation technique, and evaluated together with beads, chemical and mechanical stability in standard and stressing conditions. Beads permeability was tested towards albumin, fetuin, pyruvate, glucose, pullulan. Results demonstrated that 0.25% alginate cross-linked in 100 mM CaCl2 beads is suitable to follicle encapsulation. PMID:26791322

  14. 3D scalar model as a 4D perfect conductor limit: Dimensional reduction and variational boundary conditions

    SciTech Connect

    Edery, Ariel; Graham, Noah; MacDonald, Ilana

    2009-06-15

    Under dimensional reduction, a system in D spacetime dimensions will not necessarily yield its D-1-dimensional analog version. Among other things, this result will depend on the boundary conditions and the dimension D of the system. We investigate this question for scalar and Abelian gauge fields under boundary conditions that obey the symmetries of the action. We apply our findings to the Casimir piston, an ideal system for detecting boundary effects. Our investigation is not limited to extra dimensions and we show that the original piston scenario proposed in 2004, a toy model involving a scalar field in 3D (2+1) dimensions, can be obtained via dimensional reduction from a more realistic 4D electromagnetic (EM) system. We show that for perfect conductor conditions, a D-dimensional EM field reduces to a D-1 scalar field and not its lower-dimensional version. For Dirichlet boundary conditions, no theory is recovered under dimensional reduction and the Casimir pressure goes to zero in any dimension. This ''zero Dirichlet'' result is useful for understanding the EM case. We then identify two special systems where the lower-dimensional version is recovered in any dimension: systems with perfect magnetic conductor (PMC) and Neumann boundary conditions. We show that these two boundary conditions can be obtained from a variational procedure in which the action vanishes outside the bounded region. The fields are free to vary on the surface and have zero modes, which survive after dimensional reduction.

  15. Regional airflow and particle distribution in the lung with a 3D-1D coupled subject-specific boundary condition

    NASA Astrophysics Data System (ADS)

    Choi, Jiwoong; Yin, Youbing; Hoffman, Eric; Tawhai, Merryn; Lin, Ching-Long

    2010-11-01

    Correct prediction of regional distribution of inhaled aerosol particles is vital to improve pulmonary medicine. Physiologically consistent regional ventilations of airflow and aerosol particles are simulated with a 3D-1D coupled subject-specific boundary condition (BC). In 3D CT-resolved 7-generation airways, large eddy simulations are performed to capture detailed airflow characteristics and Lagrangian particle simulations are carried to track the particle transport and deposition. Results are compared with two traditional outlet BCs: uniform velocity and uniform pressure. Proposed BC is eligible for physiologically consistent airflow distribution in the lung, while the others are not. The regional ventilation and deposition of particles reflect the regional ventilation of airflow. In this study, two traditional BCs yield up to 98% (334%) over-prediction in lobar particle ventilation (deposition) fraction. Upper to lower particle ventilation ratios of both left and right lungs read ˜0.4 with the proposed BC, while those for the other two BCs vary with the error up to 73%.

  16. Brain damage in methylmalonic aciduria: 2-methylcitrate induces cerebral ammonium accumulation and apoptosis in 3D organotypic brain cell cultures

    PubMed Central

    2013-01-01

    Background Methylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage. Methods To explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose–response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3. Results Among the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose–response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA. Conclusion Surprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium

  17. Decrease of reactive oxygen species-related biomarkers in the tissue-mimic 3D spheroid culture of human lung cells exposed to zinc oxide nanoparticles.

    PubMed

    Kim, Eunjoo; Jeon, Won Bae; Kim, Soonhyun; Lee, Soo-Keun

    2014-05-01

    Common 2-dimensional (2D) cell cultures do not adequately represent cell-cell and cell-matrix signaling and substantially different diffusion/transport pathways. To obtain tissue-mimic information on nanoparticle toxicity from in vitro cell tests, we used a 3-dimensional (3D) culture of human lung cells (A549) prepared with elastin-like peptides modified with an arginine-glycine-aspartate motif. The 3D cells showed different cellular phenotypes, gene expression profiles, and functionalities compared to the 2D cultured cells. In gene array analysis, 3D cells displayed the induced extracellular matrix (ECM)-related biological functions such as cell-to-cell signaling and interaction, cellular function and maintenance, connective tissue development and function, molecular transport, and tissue morphology. Additionally, the expression of ECM-related molecules, such as laminin, fibronectin, and insulin-like growth factor binding protein 3 (IGFBP3), was simultaneously induced at both mRNA and protein levels. When 0.08-50 microg/ml zinc oxide nanoparticles (ZnO-NPs) were administered to 2D and 3D cells, the cell proliferation was not significantly changed. The level of molecular markers for oxidative stress, such as superoxide dismutase (SOD), Bcl-2, ATP synthase, and Complex IV (cytochrome C oxidase), was significantly reduced in 2D culture when exposed to 10 microg/ml ZnO-NPs, but no significant decrease was detected in 3D culture when exposed to the same concentration of ZnO-NPs. In conclusion, the tissue-mimic phenotype and functionality of 3D cells could be achieved through the elevated expression of ECM components. The 3D cells were expected to help to better predict the nanotoxicity of ZnO-NPs at tissue-level by increased cell-cell and cell-ECM adhesion and signaling. The tissue-mimic morphology would also be useful to simulate the diffusion/transport of the nanoparticles in vitro. PMID:24734552

  18. A Dielectric-Filled Waveguide Antenna Element for 3D Imaging Radar in High Temperature and Excessive Dust Conditions.

    PubMed

    Xu, Ding; Li, Zhiping; Chen, Xianzhong; Wang, Zhengpeng; Wu, Jianhua

    2016-01-01

    Three-dimensional information of the burden surface in high temperature and excessive dust industrial conditions has been previously hard to obtain. This paper presents a novel microstrip-fed dielectric-filled waveguide antenna element which is resistant to dust and high temperatures. A novel microstrip-to-dielectric-loaded waveguide transition was developed. A cylinder and cuboid composite structure was employed at the terminal of the antenna element, which improved the return loss performance and reduced the size. The proposed antenna element was easily integrated into a T-shape multiple-input multiple-output (MIMO) imaging radar system and tested in both the laboratory environment and real blast furnace environment. The measurement results show that the proposed antenna element works very well in industrial 3D imaging radar. PMID:27556469

  19. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.

    PubMed

    Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-Ichi; Kotani, Eiji; Hirano, Tomoko; Nakajima, Yumiko; Matsumoto, Goichi; Mori, Hajime

    2014-09-01

    Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6. PMID:25063093

  20. Optimization of Aqueous Biphasic Tumor Spheroid Microtechnology for Anti-Cancer Drug Testing in 3D Culture

    PubMed Central

    Lemmo, Stephanie; Atefi, Ehsan; Luker, Gary D.; Tavana, Hossein

    2014-01-01

    Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories. PMID:25221631

  1. 3D simulation as a tool for improving the safety culture during remediation work at Andreeva Bay.

    PubMed

    Chizhov, K; Sneve, M K; Szőke, I; Mazur, I; Mark, N K; Kudrin, I; Shandala, N; Simakov, A; Smith, G M; Krasnoschekov, A; Kosnikov, A; Kemsky, I; Kryuchkov, V

    2014-12-01

    Andreeva Bay in northwest Russia hosts one of the former coastal technical bases of the Northern Fleet. Currently, this base is designated as the Andreeva Bay branch of Northwest Center for Radioactive Waste Management (SevRAO) and is a site of temporary storage (STS) for spent nuclear fuel (SNF) and other radiological waste generated during the operation and decommissioning of nuclear submarines and ships. According to an integrated expert evaluation, this site is the most dangerous nuclear facility in northwest Russia. Environmental rehabilitation of the site is currently in progress and is supported by strong international collaboration. This paper describes how the optimization principle (ALARA) has been adopted during the planning of remediation work at the Andreeva Bay STS and how Russian-Norwegian collaboration greatly contributed to ensuring the development and maintenance of a high level safety culture during this process. More specifically, this paper describes how integration of a system, specifically designed for improving the radiological safety of workers during the remediation work at Andreeva Bay, was developed in Russia. It also outlines the 3D radiological simulation and virtual reality based systems developed in Norway that have greatly facilitated effective implementation of the ALARA principle, through supporting radiological characterisation, work planning and optimization, decision making, communication between teams and with the authorities and training of field operators. PMID:25254659

  2. Drug-releasing nano-engineered titanium implants: therapeutic efficacy in 3D cell culture model, controlled release and stability.

    PubMed

    Gulati, Karan; Kogawa, Masakazu; Prideaux, Matthew; Findlay, David M; Atkins, Gerald J; Losic, Dusan

    2016-12-01

    There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an example of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements. PMID:27612777

  3. Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

    PubMed Central

    Rodrigues, Juliany C.F.; Viana, Nathan B.; Pontes, Bruno; Pereira, Camila F.A.; Silva-Filho, Fernando C.

    2014-01-01

    Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model. PMID:24765565

  4. In situ-forming click-crosslinked gelatin based hydrogels for 3D culture of thymic epithelial cells.

    PubMed

    Truong, Vinh X; Hun, Michael L; Li, Fanyi; Chidgey, Ann P; Forsythe, John S

    2016-07-21

    Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications. PMID:27217071

  5. 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. PMID:25654124

  6. 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

  7. An Application for Cultural Heritage in Erasmus Placement. Surveys and 3d Cataloging Archaeological Finds in MÉRIDA (spain)

    NASA Astrophysics Data System (ADS)

    Barba, S.; Fiorillo, F.; Ortiz Coder, P.; D'Auria, S.; De Feo, E.

    2011-09-01

    Man has always had the need to live with his past, with its places and its artefacts. The reconstructions, the economical changes, the urbanization and its speculations have devastated whole cities, changed the faces of their historical centers, changed the relationship between the new and the old. Also the millenarian 'rest' of the archaeological findings, and therefore the respect towards those ancient civilizations, has been troubled. Our continent is rich in masterpieces that the modern man are not able to protect and pass on to the future, it is commonplace to observe that the modern `civilization' has cemented and suffocated the ancient city of Pompeii, or even worse, failed to protected it. Walking in the archaeological area of Paestum it can be noticed how just sixty years ago, no one had the slightest concern of fencing the amphitheatre and the Roman forum, or entire houses and shops, to lay a carpet of tar or simple to build constructions completely inferior compared to those majestic Greek temples. The engineers and the architects should be held responsible for this as based on their scientific and humanistic sensibility; they should bring together the man with his surroundings in the complete respects of the historical heritage. The interest in ancient began to change nearly three decades ago since it was realized that the "Cultural Heritage" is a major tourist attraction and, if properly managed and used, it can be an economical cornerstone. Today, thanks to survey and the 3D graphics, which provide powerful new tools, we are witnessing a new and real need for the conservation, cataloguing and enhancement as a way to revive our archaeological sites. As part of a major laboratory project, artefacts from the Roman period (I and II century b.C.), found in the Spanish city of Mérida, declared World Heritage by UNESCO in 1993, were acquired with a 3D laser scanner VIVID 910, and then catalogued. Based on these brief comments we wanted to direct the work

  8. Nanoparticle-mediated siRNA delivery assessed in a 3D co-culture model simulating prostate cancer bone metastasis.

    PubMed

    Fitzgerald, Kathleen A; Guo, Jianfeng; Raftery, Rosanne M; Castaño, Irene Mencía; Curtin, Caroline M; Gooding, Matt; Darcy, Raphael; O' Brien, Fergal J; O' Driscoll, Caitriona M

    2016-09-25

    siRNA has emerged as a potential therapeutic for the treatment of prostate cancer but effective delivery remains a major barrier to its clinical application. This study aimed to develop and characterise a 3D in vitro co-culture model to simulate prostate cancer bone metastasis and to assess the ability of the model to investigate nanoparticle-mediated siRNA delivery and gene knockdown. PC3 or LNCaP prostate cancer cells were co-cultured with hFOB 1.19 osteoblast cells in 2D on plastic tissue culture plates and in 3D on collagen scaffolds mimicking the bone microenvironment. To characterise the co-culture model, cell proliferation, enzyme secretion and the utility of two different gene delivery vectors to mediate siRNA uptake and gene knockdown were assessed. Cell proliferation was reduced by∼50% by day 7 in the co-culture system relative to monoculture (PC3 and LNCaP co-cultures, in 2D and 3D) and an enhanced level of MMP9 (a marker of bone metastasis) was secreted into the media (1.2-4-fold increase depending on the co-culture system). A cationic cyclodextrin gene delivery vector proved significantly less toxic in the co-culture system relative to the commercially available vector Lipofectamine 2000(®). In addition, knockdown of both the GAPDH gene (minimum 15%) and RelA subunit of the NF-κB transcription factor (minimum 20%) was achieved in 2D and 3D cell co-cultures. Results indicate that the prostate cancer-osteoblast in vitro co-culture model was more physiologically relevant vs the monoculture. This model has the potential to help improve the design and efficacy of gene delivery formulations, to more accurately predict in vivo performance and, therefore, to reduce the risk of product failure in late-stage clinical development. PMID:27492023

  9. High-Throughput 3D Screening Reveals Differences in Drug Sensitivities between Culture Models of JIMT1 Breast Cancer Cells

    PubMed Central

    Fey, Vidal; Mpindi, John-Patrick; Kleivi Sahlberg, Kristine; Kallioniemi, Olli; Perälä, Merja

    2013-01-01

    The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional monolayers on plastic. However, many cellular features are impaired in these artificial conditions, and large changes in gene expression compared to tumors have been reported. Three-dimensional cell culture models have become increasingly popular and are suggested to be better models than two-dimensional monolayers due to improved cell-to-cell contact and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput three-dimensional drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in two dimensions, in poly(2-hydroxyethyl methacrylate) induced anchorage-independent three-dimensional models, and in Matrigel three-dimensional cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating, or they were allowed to grow in three-dimensional cultures for 4 days before the drug treatment. Large variations in drug responses were observed between the models indicating that comparisons of culture model-influenced drug sensitivities cannot be made based on the effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in two-dimensional cultures, while the responses of cells grown in poly(2-hydroxyethyl methacrylate) resembled those of the two-dimensional cultures. Furthermore, comparing the gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study, we also suggest that three-dimensional cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode

  10. The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets.

    PubMed

    Nagamoto, Yasuhito; Tashiro, Katsuhisa; Takayama, Kazuo; Ohashi, Kazuo; Kawabata, Kenji; Sakurai, Fuminori; Tachibana, Masashi; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

    2012-06-01

    Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening. PMID:22445253

  11. State-of-The-Art and Applications of 3D Imaging Sensors in Industry, Cultural Heritage, Medicine, and Criminal Investigation

    PubMed Central

    Sansoni, Giovanna; Trebeschi, Marco; Docchio, Franco

    2009-01-01

    3D imaging sensors for the acquisition of three dimensional (3D) shapes have created, in recent years, a considerable degree of interest for a number of applications. The miniaturization and integration of the optical and electronic components used to build them have played a crucial role in the achievement of compactness, robustness and flexibility of the sensors. Today, several 3D sensors are available on the market, even in combination with other sensors in a “sensor fusion” approach. An importance equal to that of physical miniaturization has the portability of the measurements, via suitable interfaces, into software environments designed for their elaboration, e.g., CAD-CAM systems, virtual renders, and rapid prototyping tools. In this paper, following an overview of the state-of-art of 3D imaging sensors, a number of significant examples of their use are presented, with particular reference to industry, heritage, medicine, and criminal investigation applications. PMID:22389618

  12. State-of-The-Art and Applications of 3D Imaging Sensors in Industry, Cultural Heritage, Medicine, and Criminal Investigation.

    PubMed

    Sansoni, Giovanna; Trebeschi, Marco; Docchio, Franco

    2009-01-01

    3D imaging sensors for the acquisition of three dimensional (3D) shapes have created, in recent years, a considerable degree of interest for a number of applications. The miniaturization and integration of the optical and electronic components used to build them have played a crucial role in the achievement of compactness, robustness and flexibility of the sensors. Today, several 3D sensors are available on the market, even in combination with other sensors in a "sensor fusion" approach. An importance equal to that of physical miniaturization has the portability of the measurements, via suitable interfaces, into software environments designed for their elaboration, e.g., CAD-CAM systems, virtual renders, and rapid prototyping tools. In this paper, following an overview of the state-of-art of 3D imaging sensors, a number of significant examples of their use are presented, with particular reference to industry, heritage, medicine, and criminal investigation applications. PMID:22389618

  13. 3D analytical investigation of melting at lower mantle conditions in the laser-heated diamond anvil cel

    NASA Astrophysics Data System (ADS)

    Nabiei, F.; Cantoni, M.; Badro, J.; Dorfman, S. M.; Gaal, R.; Piet, H.; Gillet, P.

    2015-12-01

    The diamond anvil cell is a unique tool to study materials under static pressures up to several hundreds of GPa. It is possible to generate temperatures as high as several thousand degrees in the diamond anvil cell by laser heating. This allows us to achieve deep mantle conditions in the laser-heated diamond anvil cell (LHDAC). The small heated volume is surrounded by thermally conductive diamond anvils results in high temperature gradients which affect phase transformation and chemical distribution in the LH-DAC. Analytical characterization of samples in three dimensions is essential to fully understand phase assemblages and equilibrium in LHDAC. In this study we used San Carlos olivine as a starting material as a simple proxy to deep mantle composition. Three samples were melted at ~3000 K and at ~45 GPa for three different durations ranging from 1 to 6 minutes; two other samples were melted at 30 GPa and 70 GPa. All samples were then sliced by focused ion beam (FIB). From each slice, an electron image and energy dispersive X-ray (EDX) map were acquired by scanning electron microscope (SEM) in the dual beam FIB instrument. These slices were collected on one half of the heated area in each sample, from which we obtained 3D elemental and phase distribution. The other half of the heated area was used to extract a 100 nm thick section for subsequent analysis by analytical transmission electron microscopy (TEM) to obtain diffraction patterns and high resolution EDX maps. 3D reconstruction of SEM EDX results shows at least four differentiated regions in the heated area for all samples. The exact Fe and Mg compositions mentioned below are an example of the sample melted at 45 GPa for 6 minutes. The bulk of the heated are is surrounded by ferropericlase (Mg0.92, Fe0.08)O shell (Fp). Inside this shell we find a thick region of (Mg,Fe)SiO3 perovskite-structured bridgmanite (Brg) coexisting with Fp. In the center lies a Fe-rich core which is surrounded by magnesiow

  14. 3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

    NASA Astrophysics Data System (ADS)

    Meakin, J. P.; Speight, J. D.; Sheridan, R. S.; Bradshaw, A.; Harris, I. R.; Williams, A. J.; Walton, A.

    2016-08-01

    Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

  15. 3D finite element analysis of a metallic sphere scatterer comparison of first and second order vector absorbing boundary conditions

    NASA Astrophysics Data System (ADS)

    Kanellopoulos, V. N.; Webb, J. P.

    1993-03-01

    A 3D vector analysis of plane wave scattering by a metallic sphere using finite elements and Absorbing Boundary Conditions (ABCs) is presented. The ABCs are applied on the outer surface that truncates the infinitely extending domain. Mixed order curvilinear covariantprojection elements are used to avoid spurious corruptions. The second order ABC is superior to the first at no extra computational cost. The errors due to incomplete absorption decrease as the outer surface is moved further away from the scatterer. An error of about 1% in near-field values was obtained with the second order ABC, when the outer surface was less than half a wavelength from the scatterer. Une analyse tridimensionnelle vectorielle de la diffusion d'onde plane sur une sphère métallique utilisant des éléments finis et des Conditions aux Limites Absorbantes (CLA) est présentée. Les CLA sont appliquées sur la surface exteme tronquant le domaine s'étendant à l'infini. Des éléments curvilignes mixtes utilisant des projections covariantes sont utilisés pour éviter des solutions parasites. La CLA de second ordre est supérieure à celle de premier ordre sans effort de calcul additionnel. Les erreurs dues à l'absorption incomplète décroissent à mesure que l'on déplace la surface externe à une distance croissante du diffuseur. Un taux d'erreur d'environ 1 % dans les valeurs du champ proche a été obtenu avec les CLA de second ordre lorsque la surface externe était placée à une distance inférieure à une demi-longueur de la source de diffusion.

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

    SciTech Connect

    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{sup 5}-10{sup 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{sub 50}) using commercially available drugs which further correlated well with published in vivo LD{sub 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. - Graphical abstract: Display Omitted Highlights: > A porous support disc design to support the culture of desired cells in 3D hydrogels. > Demonstrated the co-culture of two cell types within standard cell-culture plates. > A scalable, low cost approach to toxicity screening involving

  17. Printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3D cell culture.

    PubMed

    Müller, Michael; Becher, Jana; Schnabelrauch, Matthias; Zenobi-Wong, Marcy

    2013-01-01

    Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting(1-4) (extrusion, dip pen and soft lithography), contactless bioprinting(5-7) (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization(8). It can be used for many applications such as tissue engineering(9-13), biosensor microfabrication(14-16) and as a tool to answer basic biological questions such as influences of co-culturing of different cell types(17). Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches. Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 °C and a solid above its gelation temperature ~20 °C for 24.5% w/v solutions(18). This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an

  18. Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

    PubMed Central

    Müller, Michael; Becher, Jana; Schnabelrauch, Matthias; Zenobi-Wong, Marcy

    2013-01-01

    Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches. Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 °C and a solid above its gelation temperature ~20 °C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi

  19. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

    PubMed

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6) cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia. PMID:23823334

  20. Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells

    PubMed Central

    Gaberman, Elena; Pinzur, Lena; Levdansky, Lilia; Tsirlin, Maria; Netzer, Nir; Aberman, Zami; Gorodetsky, Raphael

    2013-01-01

    Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×106 cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective “off the shelf” therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia. PMID:23823334

  1. Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

    SciTech Connect

    Gu, Angel; Tsark, Walter; Holmes, Kathryn V.; Shively, John E.

    2009-06-10

    CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (- 8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as - 5 to - 3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased

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

    PubMed Central

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

    2013-01-01

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

  3. Heterogeneous reactive transport under unsaturated transient conditions characterized by 3D electrical resistivity tomography and advanced lysimeter methods

    NASA Astrophysics Data System (ADS)

    Wehrer, Markus; Slater, Lee

    2015-04-01

    flow fraction was observed to be independent of precipitation rate. This suggests the presence of a fingering process driven by textural heterogeneities. As a consequence, preferential transport of the conservative and the reactive tracer also occurred. We found that 3D ERT can serve to quantitatively characterize shape measures of both tracer breakthroughs and water content dynamics. In particular, shape measures influenced by the advective propagation of the tracer peak, like mean velocity and normalized first central moment, are highly correlated between ERT data and validation data (consisting of tracer measurements in seepage water samples). Using shape measures proved to be advantageous over interpretation of ERT data with spatially uncertain petrophysical functions for the characterization of heterogeneous flow and transport. Consequently, for future applications of ERT in soil hydrological modeling, the use of temporal moments is recommended.

  4. Osteogenic potential of human adipose-tissue-derived mesenchymal stromal cells cultured on 3D-printed porous structured titanium.

    PubMed

    Lewallen, Eric A; Jones, Dakota L; Dudakovic, Amel; Thaler, Roman; Paradise, Christopher R; Kremers, Hilal M; Abdel, Matthew P; Kakar, Sanjeev; Dietz, Allan B; Cohen, Robert C; Lewallen, David G; van Wijnen, Andre J

    2016-05-01

    Integration of porous metal prosthetics, which restore form and function of irreversibly damaged joints, into remaining healthy bone is critical for implant success. We investigated the biological properties of adipose-tissue-derived mesenchymal stromal/stem cells (AMSCs) and addressed their potential to alter the in vitro microenvironment of implants. We employed human AMSCs as a practical source for musculoskeletal applications because these cells can be obtained in large quantities, are multipotent, and have trophic paracrine functions. AMSCs were cultured on surgical-grade porous titanium disks as a model for orthopedic implants. We monitored cell/substrate attachment, cell proliferation, multipotency, and differentiation phenotypes of AMSCs upon osteogenic induction. High-resolution scanning electron microscopy and histology revealed that AMSCs adhere to the porous metallic surface. Compared to standard tissue culture plastic, AMSCs grown in the porous titanium microenvironment showed differences in temporal expression for genes involved in cell cycle progression (CCNB2, HIST2H4), extracellular matrix production (COL1A1, COL3A1), mesenchymal lineage identity (ACTA2, CD248, CD44), osteoblastic transcription factors (DLX3, DLX5, ID3), and epigenetic regulators (EZH1, EZH2). We conclude that metal orthopedic implants can be effectively seeded with clinical-grade stem/stromal cells to create a pre-conditioned implant. PMID:26774799

  5. Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications

    PubMed Central

    Nagymihály, Richárd; Josifovska, Natasha; Andjelic, Sofija; Veréb, Zoltán; Facskó, Andrea; Moe, Morten C.; Petrovski, Goran

    2015-01-01

    Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation. PMID:26580800

  6. Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications.

    PubMed

    Szabó, Dóra Júlia; Noer, Agate; Nagymihály, Richárd; Josifovska, Natasha; Andjelic, Sofija; Veréb, Zoltán; Facskó, Andrea; Moe, Morten C; Petrovski, Goran

    2015-01-01

    Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation. PMID:26580800

  7. Correlation between lack of norovirus replication and histo-blood group antigen expression in 3D-intestinal epithelial cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Noroviruses (NoV) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. One publication utilizing a 3-dimensional (3D) intestinal model derived from Int407 cells reported NoV replication and extensive cytopathi...

  8. Mechanism for generating stagnant slabs in 3-D spherical mantle convection models at Earth-like conditions

    NASA Astrophysics Data System (ADS)

    Yanagisawa, Takatoshi; Yamagishi, Yasuko; Hamano, Yozo; Stegman, Dave R.; Suetsugu, Daisuke; Bina, Craig; Inoue, Toru; Wiens, Douglas; Jellinek, Mark

    2010-11-01

    Seismic tomography reveals the natural mode of convection in the Earth is whole mantle with subducted slabs clearly seen as continuous features into the lower mantle. However, simultaneously existing alongside these deep slabs are stagnant slabs which are, if only temporarily, trapped in the upper mantle. Previous numerical models of mantle convection have observed a range of behavior for slabs in the transition zone depending on viscosity stratification and mineral phase transitions, but typically only exhibit flat-lying slabs when mantle convection is layered or trench migration is imposed. We use 3-D spherical models of mantle convection which range up to Earth-like conditions in Rayleigh number to systematically investigate three effects on mantle dynamics: (1) the mineral phase transitions, (2) a strongly temperature-dependent viscosity with plastic yielding at shallow depth, and (3) a viscosity increase in the lower mantle. First a regime diagram is constructed for isoviscous models over a wide range of Rayleigh number and Clapeyron slope for which the convective mode is determined. It agrees very well with previous results from 2-D simulations by Christensen and Yuen (1985), suggesting present-day Earth is in the intermittent convection mode rather than layered or strictly whole mantle. Two calculations at Earth-like conditions (Ra and RaH = 2 í 107 and 5 í 108, respectively) which include effects (2) and (3) are produced with and without the effect of the mineral phase transitions. The first calculation (without the phase transition) successfully produces plate-like behavior with a long wavelength structure and surface heat flow similar to Earth's value. While the observed convective flow pattern in the lower mantle is broader compared to isoviscous models, it basically shows the behavior of whole mantle convection, and does not exhibit any slab flattening at the viscosity increase at 660 km depth. The second calculation which includes the phase

  9. Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study

    PubMed Central

    Moscato, Stefania; Ronca, Francesca; Campani, Daniela; Danti, Serena

    2015-01-01

    It has been demonstrated that three-dimensional (3D) cell culture models represent fundamental tools for the comprehension of cellular phenomena both for normal and cancerous tissues. Indeed, the microenvironment affects the cellular behavior as well as the response to drugs. In this study, we performed a morphological analysis on a hepatocarcinoma cell line, HepG2, grown for 24 days inside a bioartificial hydrogel composed of poly(vinyl alcohol) (PVA) and gelatin (G) to model a hepatocellular carcinoma (HCC) in 3D. Morphological features of PVA/G hydrogels were investigated, resulting to mimic the trabecular structure of liver parenchyma. A histologic analysis comparing the 3D models with HepG2 cell monolayers and tumor specimens was performed. In the 3D setting, HepG2 cells were viable and formed large cellular aggregates showing different morphotypes with zonal distribution. Furthermore, β-actin and α5β1 integrin revealed a morphotype-related expression; in particular, the frontline cells were characterized by a strong immunopositivity on a side border of their membrane, thus suggesting the formation of lamellipodia-like structures apt for migration. Based on these results, we propose PVA/G hydrogels as valuable substrates to develop a long term 3D HCC model that can be used to investigate important aspects of tumor biology related to migration phenomena. PMID:25590431

  10. A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC).

    PubMed

    Alessandri, Kevin; Feyeux, Maxime; Gurchenkov, Basile; Delgado, Christophe; Trushko, Anastasiya; Krause, Karl-Heinz; Vignjević, Daniela; Nassoy, Pierre; Roux, Aurélien

    2016-04-26

    We present here a microfluidic device that generates sub-millimetric hollow hydrogel spheres, encapsulating cells and coated internally with a layer of reconstituted extracellular matrix (ECM) of a few microns thick. The spherical capsules, composed of alginate hydrogel, originate from the spontaneous instability of a multi-layered jet formed by co-extrusion using a coaxial flow device. We provide a simple design to manufacture this device using a DLP (digital light processing) 3D printer. Then, we demonstrate how the inner wall of the capsules can be decorated with a continuous ECM layer that is anchored to the alginate gel and mimics the basal membrane of a cellular niche. Finally, we used this approach to encapsulate human Neural Stem Cells (hNSC) derived from human Induced Pluripotent Stem Cells (hIPSC), which were further differentiated into neurons within the capsules with negligible loss of viability. Altogether, we show that these capsules may serve as cell micro-containers compatible with complex cell culture conditions and applications. These developments widen the field of research and biomedical applications of the cell encapsulation technology. PMID:27025278

  11. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

    PubMed Central

    Schmal, Olga; Seifert, Jan; Schäffer, Tilman E.; Walter, Christina B.; Aicher, Wilhelm K.; Klein, Gerd

    2016-01-01

    Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard. PMID:26839560

  12. Differential Radiosensitizing Effect of Valproic Acid in Differentiation Versus Self-Renewal Promoting Culture Conditions

    SciTech Connect

    Debeb, Bisrat G.; Xu Wei; Mok, Henry; Li Li; Robertson, Fredika; Ueno, Naoto T.; Reuben, Jim; Lucci, Anthony; Cristofanilli, Massimo; Woodward, Wendy A.

    2010-03-01

    Purpose: It has been shown that valproic acid (VA) enhances the proliferation and self-renewal of normal hematopoietic stem cells and that breast cancer stem/progenitor cells can be resistant to radiation. From these data, we hypothesized that VA would fail to radiosensitize breast cancer stem/progenitor cells grown to three-dimensional (3D) mammospheres. Methods and Materials: We used the MCF7 breast cancer cell line grown under stem cell-promoting culture conditions (3D mammosphere) and standard nonstem cell monolayer culture conditions (two-dimensional) to examine the effect of pretreatment with VA on radiation sensitivity in clonogenic survival assays and on the expression of embryonic stem cell transcription factors. Results: 3D-cultured MCF-7 cells expressed higher levels of Oct4, Nanog, and Sox2. The 3D passage enriched self-renewal and increased radioresistance in the 3D mammosphere formation assays. VA radiosensitized adherent cells but radioprotected 3D cells in single-fraction clonogenic assays. Moreover, fractionated radiation sensitized VA-treated adherent MCF7 cells but did not have a significant effect on VA-treated single cells grown to mammospheres. Conclusion: We have concluded that VA might preferentially radiosensitize differentiated cells compared with those expressing stem cell surrogates and that stem cell-promoting culture is a useful tool for in vitro evaluation of novel cancer therapeutic agents and radiosensitizers.

  13. 3D micro-XRF for cultural heritage objects: new analysis strategies for the investigation of the Dead Sea Scrolls.

    PubMed

    Mantouvalou, Ioanna; Wolff, Timo; Hahn, Oliver; Rabin, Ira; Lühl, Lars; Pagels, Marcel; Malzer, Wolfgang; Kanngiesser, Birgit

    2011-08-15

    A combination of 3D micro X-ray fluorescence spectroscopy (3D micro-XRF) and micro-XRF was utilized for the investigation of a small collection of highly heterogeneous, partly degraded Dead Sea Scroll parchment samples from known excavation sites. The quantitative combination of the two techniques proves to be suitable for the identification of reliable marker elements which may be used for classification and provenance studies. With 3D micro-XRF, the three-dimensional nature, i.e. the depth-resolved elemental composition as well as density variations, of the samples was investigated and bromine could be identified as a suitable marker element. It is shown through a comparison of quantitative and semiquantitative values for the bromine content derived using both techniques that, for elements which are homogeneously distributed in the sample matrix, quantification with micro-XRF using a one-layer model is feasible. Thus, the possibility for routine provenance studies using portable micro-XRF instrumentation on a vast amount of samples, even on site, is obtained through this work. PMID:21711051

  14. Smad signal pathway regulates angiogenesis via endothelial cell in an adipose-derived stromal cell/endothelial cell co-culture, 3D gel model.

    PubMed

    Lin, Shiyu; Xie, Jing; Gong, Tao; Shi, Sirong; Zhang, Tao; Fu, Na; Lin, Yunfeng

    2016-01-01

    Co-implantation of adipose-derived stromal cells (ASCs) and endothelial cells (ECs) can markedly expedite the formation of functional microvascular beds and provides possible methods for cell-based revascularization therapies to treat various diseases. Furthermore, we investigated the role of TGFβ/Smad signaling pathway for angiogenesis in a three-dimensional (3D) collagen gel model established in vitro with co-culture between ASCs and ECs. We found that angiogenesis was attenuated in the co-culture gels after inhibition of ALK5/Smad2/3 with SB431542. Genes coding for VEGF-A, VEGF-B, VE-ca, FGF-1, PDGF, BMP-4, and BMP-7 were significantly reduced in both mono-cultured and co-cultured ECs. Furthermore, the decrease in co-cultured ECs was prominent relative to mono-cultured ECs. Taken together, these findings suggest that in the co-culture between ASCs and ECs, TGFβ/Smad signal pathway regulates angiogenesis via ECs; moreover, the findings that the co-cultured ECs were regulated more significantly than mono-cultured ECs suggest that suppression of Smad signal pathway may regulate the paracrine secretion of ASCs to further modulate angiogenesis of ECs. PMID:26694166

  15. Audio-Visual Perception of 3D Cinematography: An fMRI Study Using Condition-Based and Computation-Based Analyses

    PubMed Central

    Ogawa, Akitoshi; Bordier, Cecile; Macaluso, Emiliano

    2013-01-01

    The use of naturalistic stimuli to probe sensory functions in the human brain is gaining increasing interest. Previous imaging studies examined brain activity associated with the processing of cinematographic material using both standard “condition-based” designs, as well as “computational” methods based on the extraction of time-varying features of the stimuli (e.g. motion). Here, we exploited both approaches to investigate the neural correlates of complex visual and auditory spatial signals in cinematography. In the first experiment, the participants watched a piece of a commercial movie presented in four blocked conditions: 3D vision with surround sounds (3D-Surround), 3D with monaural sound (3D-Mono), 2D-Surround, and 2D-Mono. In the second experiment, they watched two different segments of the movie both presented continuously in 3D-Surround. The blocked presentation served for standard condition-based analyses, while all datasets were submitted to computation-based analyses. The latter assessed where activity co-varied with visual disparity signals and the complexity of auditory multi-sources signals. The blocked analyses associated 3D viewing with the activation of the dorsal and lateral occipital cortex and superior parietal lobule, while the surround sounds activated the superior and middle temporal gyri (S/MTG). The computation-based analyses revealed the effects of absolute disparity in dorsal occipital and posterior parietal cortices and of disparity gradients in the posterior middle temporal gyrus plus the inferior frontal gyrus. The complexity of the surround sounds was associated with activity in specific sub-regions of S/MTG, even after accounting for changes of sound intensity. These results demonstrate that the processing of naturalistic audio-visual signals entails an extensive set of visual and auditory areas, and that computation-based analyses can track the contribution of complex spatial aspects characterizing such life-like stimuli

  16. Audio-visual perception of 3D cinematography: an fMRI study using condition-based and computation-based analyses.

    PubMed

    Ogawa, Akitoshi; Bordier, Cecile; Macaluso, Emiliano

    2013-01-01

    The use of naturalistic stimuli to probe sensory functions in the human brain is gaining increasing interest. Previous imaging studies examined brain activity associated with the processing of cinematographic material using both standard "condition-based" designs, as well as "computational" methods based on the extraction of time-varying features of the stimuli (e.g. motion). Here, we exploited both approaches to investigate the neural correlates of complex visual and auditory spatial signals in cinematography. In the first experiment, the participants watched a piece of a commercial movie presented in four blocked conditions: 3D vision with surround sounds (3D-Surround), 3D with monaural sound (3D-Mono), 2D-Surround, and 2D-Mono. In the second experiment, they watched two different segments of the movie both presented continuously in 3D-Surround. The blocked presentation served for standard condition-based analyses, while all datasets were submitted to computation-based analyses. The latter assessed where activity co-varied with visual disparity signals and the complexity of auditory multi-sources signals. The blocked analyses associated 3D viewing with the activation of the dorsal and lateral occipital cortex and superior parietal lobule, while the surround sounds activated the superior and middle temporal gyri (S/MTG). The computation-based analyses revealed the effects of absolute disparity in dorsal occipital and posterior parietal cortices and of disparity gradients in the posterior middle temporal gyrus plus the inferior frontal gyrus. The complexity of the surround sounds was associated with activity in specific sub-regions of S/MTG, even after accounting for changes of sound intensity. These results demonstrate that the processing of naturalistic audio-visual signals entails an extensive set of visual and auditory areas, and that computation-based analyses can track the contribution of complex spatial aspects characterizing such life-like stimuli. PMID

  17. Sub-100 nm biodegradable nanoparticles: in vitro release features and toxicity testing in 2D and 3D cell cultures

    NASA Astrophysics Data System (ADS)

    Biondi, Marco; Guarnieri, Daniela; Yu, Hui; Belli, Valentina; Netti, Paolo Antonio

    2013-02-01

    A big challenge in tumor targeting by nanoparticles (NPs), taking advantage of the enhanced permeability and retention effect, is the fabrication of small size devices for enhanced tumor penetration, which is considered fundamental to improve chemotherapy efficacy. The purposes of this study are (i) to engineer the formulation of doxorubicin-loaded poly(d,l-lactic-co-glycolic acid) (PLGA)-block-poly(ethylene glycol) (PEG) NPs to obtain <100 nm devices and (ii) to translate standard 2D cytotoxicity studies to 3D collagen systems in which an initial step gradient of the NPs is present. Doxorubicin release can be prolonged for days to weeks depending on the NP formulation and the pH of the release medium. Sub-100 nm NPs are effectively internalized by HeLa cells in 2D and are less cytotoxic than free doxorubicin. In 3D, <100 nm NPs are significantly more toxic than larger ones towards HeLa cells, and the cell death rate is affected by the contributions of drug release and device transport through collagen. Thus, the reduction of NP size is a fundamental feature from both a technological and a biological point of view and must be properly engineered to optimize the tumor response to the NPs.

  18. 3D cell culture and osteogenic differentiation of human bone marrow stromal cells plated onto jet-sprayed or electrospun micro-fiber scaffolds.

    PubMed

    Brennan, Meadhbh Á; Renaud, Audrey; Gamblin, Anne-Laure; D'Arros, Cyril; Nedellec, Steven; Trichet, Valerie; Layrolle, Pierre

    2015-08-01

    A major limitation of the 2D culture systems is that they fail to recapitulate the in vivo 3D cellular microenvironment whereby cell-cell and cell-extracellular matrix (ECM) interactions occur. In this paper, a biomaterial scaffold that mimics the structure of collagen fibers was produced by jet-spraying. This micro-fiber polycaprolactone (PCL) scaffold was evaluated for 3D culture of human bone marrow mesenchymal stromal cells (MSCs) in comparison with a commercially available electrospun scaffold. The jet-sprayed scaffolds had larger pore diameters, greater porosity, smaller diameter fibers, and more heterogeneous fiber diameter size distribution compared to the electrospun scaffolds. Cells on jet-sprayed constructs exhibited spread morphology with abundant cytoskeleton staining, whereas MSCs on electrospun scaffolds appeared less extended with fewer actin filaments. MSC proliferation and cell infiltration occurred at a faster rate on jet-sprayed compared to electrospun scaffolds. Osteogenic differentiation of MSCs and ECM production as measured by ALP, collagen and calcium deposition was superior on jet-sprayed compared to electrospun scaffolds. The jet-sprayed scaffold which mimics the native ECM and permits homogeneous cell infiltration is important for 3D in vitro applications such as bone cellular interaction studies or drug testing, as well as bone tissue engineering strategies. PMID:26238732

  19. One-Step Microfluidic Generation of Pre-Hatching Embryo-Like Core-Shell Microcapsules for Miniaturized 3D Culture of Pluripotent Stem Cells

    PubMed Central

    Agarwal, Pranay; Zhao, Shuting; Bielecki, Peter; Rao, Wei; Choi, Jung K.; Zhao, Yi; Yu, Jianhua; Zhang, Wujie; He, Xiaoming

    2013-01-01

    A novel core-shell microcapsule system is developed in this study to mimic the miniaturized 3D architecture of pre-hatching embryos with an aqueous liquid core of embryonic cells and a hydrogel-shell of zona pellucida. This is done by microfabricating a non-planar microfluidic flow-focusing device that enables one-step generation of microcapsules with an alginate hydrogel shell and an aqueous liquid core of cells from two aqueous fluids. Mouse embryonic stem (ES) cells encapsulated in the liquid core are found to survive well (> 92 %). Moreover, ~ 20 ES cells in the core can proliferate to form a single ES cell aggregate in each microcapsule within 7 days while at least a few hundred cells are usually needed by the commonly used hanging-drop method to form an embryoid body (EB) in each hanging drop. Quantitative RT-PCR analyses show significantly higher expression of pluripotency marker genes in the 3D aggregated ES cells compared to the cells under 2D culture. The aggregated ES cells can be efficiently differentiated into beating cardiomyocytes using a small molecule (cardiogenol C) without complex combination of multiple growth factors. Taken together, the novel 3D microfluidic and pre-hatching embryo-like microcapsule systems are of importance to facilitate in vitro culture of pluripotent stem cells for their ever-increasing use in modern cell-based medicine. PMID:24113543

  20. Wnt5a-mediating neurogenesis of human adipose tissue-derived stem cells in a 3D microfluidic cell culture system.

    PubMed

    Choi, Jeein; Kim, Sohyeun; Jung, Jinsun; Lim, Youngbin; Kang, Kyungsun; Park, Seungsu; Kang, Sookyung

    2011-10-01

    In stem cell biology, cell plasticity refers to the ability of stem cells to differentiate into a variety of cell lineages. Recently, cell plasticity has been used to refer to the ability of a given cell type to reversibly de-differentiate, re-differentiate, or transdifferentiate in response to specific stimuli. These processes are regulated by multiple intracellular and extracellular growth and differentiation factors, including low oxygen. Our recent study showed that 3D microfluidic cell culture induces activation of the Wnt5A/β-catenin signaling pathway in hATSCs (human Adipose Tissue-derived Stem Cells). This resulted in self renewal and transdifferentiation of hATSCs into neurons. To improve neurogenic potency of hATSCs in response to low oxygen and other unknown physical factors, we developed a gel-free 3D microfluidic cell culture system (3D-μFCCS). The functional structure was developed for the immobilization of 3D multi-cellular aggregates in a microfluidic channel without the use of a matrix on the chip. Growth of hATSCs neurosphere grown on a chip was higher than the growth of control cells grown in a culture dish. Induction of differentiation in the Chip system resulted in a significant increase in the induction of neuronal-like cell structures and the presentation of TuJ or NF160 positive long neuritis compared to control cells after active migration from the center of the microfluidic channel layer to the outside of the microfluidic channel layer. We also observed that the chip neurogenesis system induced a significantly higher level of GABA secreting neurons and, in addition, almost 60% of cells were GABA + cells. Finally, we observed that 1 month of after the transplantation of each cell type in a mouse SCI lesion, chip cultured and neuronal differentiated hATSCs exhibited the ability to effectively transdifferentiate into NF160 + motor neurons at a high ratio. Interestingly, our CHIP/PCR analysis revealed that HIF1α-induced hATSCs neurogenesis

  1. Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer.

    PubMed

    Augustine, Tanya N; Dix-Peek, Thérèse; Duarte, Raquel; Candy, Geoffrey P

    2015-11-01

    Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell-cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines. PMID:26215372

  2. Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition.

    PubMed

    Leight, Jennifer L; Tokuda, Emi Y; Jones, Caitlin E; Lin, Austin J; Anseth, Kristi S

    2015-04-28

    Matrix metalloproteinases (MMPs) are important for many different types of cancer-related processes, including metastasis. Understanding the functional impact of changes in MMP activity during cancer treatment is an important facet not typically evaluated as part of preclinical research. With MMP activity being a critical component of the metastatic cascade, we designed a 3D hydrogel system to probe whether pharmacological inhibition affected human melanoma cell proteolytic activity; metastatic melanoma is a highly aggressive and drug-resistant form of skin cancer. The relationship between MMP activity and drug treatment is unknown, and therefore we used an in situ fluorogenic MMP sensor peptide to determine how drug treatment affects melanoma cell MMP activity in three dimensions. We encapsulated melanoma cells from varying stages of progression within PEG-based hydrogels to examine the relationship between drug treatment and MMP activity. From these results, a metastatic melanoma cell line (A375) and two inhibitors that inhibit RAF (PLX4032 and sorafenib) were studied further to determine whether changes in MMP activity led to a functional change in cell behavior. A375 cells exhibited increased MMP activity despite an overall decrease in metabolic activity with PLX4032 treatment. The changes in proteolytic activity correlated with increased cell elongation and increased single-cell migration. In contrast, sorafenib did not alter MMP activity or cell motility, showing that the changes induced by PLX4032 were not a universal response to small-molecule inhibition. Therefore, we argue the importance of studying MMP activity with drug treatment and its possible implications for unwanted side effects. PMID:25870264

  3. Reduced cytotoxicity and enhanced bioactivity of cationic antimicrobial peptides liposomes in cell cultures and 3D epidermis model against HSV.

    PubMed

    Ron-Doitch, Sapir; Sawodny, Beate; Kühbacher, Andreas; David, Mirjam M Nordling; Samanta, Ayan; Phopase, Jaywant; Burger-Kentischer, Anke; Griffith, May; Golomb, Gershon; Rupp, Steffen

    2016-05-10

    Cationic antimicrobial peptides (AMPs) are part of the innate immunity, and act against a wide variety of pathogenic microorganisms by perturbation of the microorganism's plasma membrane. Although attractive for clinical applications, these agents suffer from limited stability and activity in vivo, as well as non-specific interaction with host biological membranes, leading to cytotoxic adverse effects. We hypothesized that encapsulation of AMPs within liposomes could result in reduced cytotoxicity, and with enhanced stability as well as bioactivity against herpes simplex virus 1 (HSV-1). We formulated nano-sized liposomal formulations of LL-37 and indolicidin, and their physicochemical properties, cellular uptake, in vitro cytotoxicity and antiviral efficacy have been determined. Lower cytotoxicity of LL-37 liposomes was found in comparison to indolicidin liposomes attributed to the superior physicochemical properties, and to the different degree of interaction with the liposomal membrane. The disc-like shaped LL-37 liposomes (106.8±10.1nm, shelf-life stability of >1year) were taken up more rapidly and to a significantly higher extent than the free peptide by human keratinocyte cell line (HaCaT), remained intact within the cells, followed by release of the active peptide within the cytoplasm and migration of the vesicles' lipids to the plasma membrane. LL-37 liposomes were found significantly less toxic than both the free agent and liposomal indolicidin. In the new 3D epidermis model (immortalized primary keratinocytes) liposomal LL-37 treatment (>20μM), but not free LL-37, efficiently protected the epidermis, inhibiting HSV-1 infection. This positive antiviral effect was obtained with no cytotoxicity even at very high concentrations (400μM). Thus, the antiviral activity of encapsulated LL-37 was significantly improved, expanding its therapeutic window. Liposomal LL-37 appears to be a promising delivery system for HSV therapy. PMID:27012977

  4. Enhanced Metabolizing Activity of Human ES Cell-Derived Hepatocytes Using a 3D Culture System with Repeated Exposures to Xenobiotics.

    PubMed

    Kim, Jong Hyun; Jang, Yu Jin; An, Su Yeon; Son, Jeongsang; Lee, Jaehun; Lee, Gyunggyu; Park, Ji Young; Park, Han-Jin; Hwang, Dong-Youn; Kim, Jong-Hoon; Han, Jiyou

    2015-09-01

    Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the cell-based therapy of liver disease and the development of effective in vitro toxicity screening tools. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized hepatic differentiation protocol, which produces >90% (BGO1 and CHA15) albumin-positive HLCs with no purification process from human embryonic stem cell lines. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating 3D spheroidal aggregate of HLCs, compared with 2D HLCs. The 3D differentiation method increased expression of nuclear receptors (NRs) that regulate the proper expression of key hepatic enzymes. Furthermore, significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids, compared with 2D HLCs. HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated further functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic metabolizing ability of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models. PMID:26089346

  5. Capturing 3D resistivity of semi-arid karstic subsurface in varying moisture conditions using a wireless sensor network

    NASA Astrophysics Data System (ADS)

    Barnhart, K.; Oden, C. P.

    2012-12-01

    The dissolution of soluble bedrock results in surface and subterranean karst channels, which comprise 7-10% of the dry earth's surface. Karst serves as a preferential conduit to focus surface and subsurface water but it is difficult to exploit as a water resource or protect from pollution because of irregular structure and nonlinear hydrodynamic behavior. Geophysical characterization of karst commonly employs resistivity and seismic methods, but difficulties arise due to low resistivity contrast in arid environments and insufficient resolution of complex heterogeneous structures. To help reduce these difficulties, we employ a state-of-the-art wireless geophysical sensor array, which combines low-power radio telemetry and solar energy harvesting to enable long-term in-situ monitoring. The wireless aspect removes topological constraints common with standard wired resistivity equipment, which facilitates better coverage and/or sensor density to help improve aspect ratio and resolution. Continuous in-situ deployment allows data to be recorded according to nature's time scale; measurements are made during infrequent precipitation events which can increase resistivity contrast. The array is coordinated by a smart wireless bridge that continuously monitors local soil moisture content to detect when precipitation occurs, schedules resistivity surveys, and periodically relays data to the cloud via 3G cellular service. Traditional 2/3D gravity and seismic reflection surveys have also been conducted to clarify and corroborate results.

  6. Bioprinting of 3D hydrogels.

    PubMed

    Stanton, M M; Samitier, J; Sánchez, S

    2015-08-01

    Three-dimensional (3D) bioprinting has recently emerged as an extension of 3D material printing, by using biocompatible or cellular components to build structures in an additive, layer-by-layer methodology for encapsulation and culture of cells. These 3D systems allow for cell culture in a suspension for formation of highly organized tissue or controlled spatial orientation of cell environments. The in vitro 3D cellular environments simulate the complexity of an in vivo environment and natural extracellular matrices (ECM). This paper will focus on bioprinting utilizing hydrogels as 3D scaffolds. Hydrogels are advantageous for cell culture as they are highly permeable to cell culture media, nutrients, and waste products generated during metabolic cell processes. They have the ability to be fabricated in customized shapes with various material properties with dimensions at the micron scale. 3D hydrogels are a reliable method for biocompatible 3D printing and have applications in tissue engineering, drug screening, and organ on a chip models. PMID:26066320

  7. Development of transferrin targeted NCL-240 micelles and their evaluation using in-vitro 3D cancer cell culture (spheroid) models

    NASA Astrophysics Data System (ADS)

    Nagelli, Srikar Goud

    The main objective of this project was to develop targeted micellar delivery systems of a novel cytotoxic drug (NCL-240; a second generation DM-PIT-1 analog) and to evaluate their efficacy using optimized 3D cell culture spheroid models. Spheroids were optimized for several cancer cell lines using a range of techniques such as non-adhesive liquid overlay method, hanging drop method, and co-culturing. Transferrin (Tf)-conjugated NCL-240 micelles were prepared with varying Tf amounts and their cytotoxicities were evaluated using the optimized spheroid models. The uptake and penetration of the formulations were also studied using confocal microscopy. The results indicated that the concentration of NCL-240 micelles required to achieve the same cytotoxicity was relatively higher in spheroids compared to the monolayers. Also, In NCI-ADR-RES, Tf-targeted NCL-240 micelles were shown to have a significant increase in cytotoxicity compared to untargeted NCL-240 micelles. Even the penetration and uptake studies indicated that targeting improves the uptake and penetration of formulations. However, in U87-MG spheroids, there was a significant difference in cell viability among micelles compared to free drug but no significant benefit due to targeting was observed. The same formulations penetrated lesser in U87-MG spheroids compared to NCI-ADR-RES spheroids. This study thereby emphasizes the importance of drug screening in spheroid models as the penetration dynamics are varying from cell line to cell line because of the 3D structure.

  8. 3D Porous Calcium-Alginate Scaffolds Cell Culture System Improved Human Osteoblast Cell Clusters for Cell Therapy

    PubMed Central

    Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

    2015-01-01

    Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects. PMID:25825603

  9. Future Research Challenges for a Computer-Based Interpretative 3D Reconstruction of Cultural Heritage - A German Community's View

    NASA Astrophysics Data System (ADS)

    Münster, S.; Kuroczyński, P.; Pfarr-Harfst, M.; Grellert, M.; Lengyel, D.

    2015-08-01

    The workgroup for Digital Reconstruction of the Digital Humanities in the German-speaking area association (Digital Humanities im deutschsprachigen Raum e.V.) was founded in 2014 as cross-disciplinary scientific society dealing with all aspects of digital reconstruction of cultural heritage and currently involves more than 40 German researchers. Moreover, the workgroup is dedicated to synchronise and foster methodological research for these topics. As one preliminary result a memorandum was created to name urgent research challenges and prospects in a condensed way and assemble a research agenda which could propose demands for further research and development activities within the next years. The version presented within this paper was originally created as a contribution to the so-called agenda development process initiated by the German Federal Ministry of Education and Research (BMBF) in 2014 and has been amended during a joint meeting of the digital reconstruction workgroup in November 2014.

  10. Evaluation of the cytotoxic effects of humid lightweight coal ash derived from the disposal of waste on normal human keratinocyte and endothelial cell lines in 2-D and 3-D culture.

    PubMed

    Scanarotti, Chiara; Vernazza, Stefania; Brignone, Massimiliano; Danailova, Jenia; Pronzato, Maria A; Bassi, Anna M

    2013-12-01

    The presence of waste in the environment has frequently been indicated as a significant risk to human health. Therefore, landfill sites and the disposal of urban solid and non-hazardous waste by incineration are subject to much environmental monitoring, in addition to the regulations already in place. However, little action has been taken, and consequently no specific legislation exists, in relation to the assessment of the real biological risk of various substances, including chemical mixtures and ashes, derived from the incineration processes. This study assessed the cytotoxic potential of humid lightweight coal ash (LA) derived from incineration processes and waste management, on two cell lines: NCTC 2544 normal human keratinocytes and HECV endothelial cells. To reach this goal and to assess more-realistic methods for animal replacement, we employed different in vitro experimental approaches: acute and longer exposure to LA, by direct and indirect contact (0-2mg/ml and 16mg, respectively), both in 2-D and 3-D cultures. In 2-D HECV cultures, we observed a decrease in the viability index, but only during direct contact with LA doses higher than 0.1mg/ml. Moreover, some striking differences in cytotoxicity were observed between the 2-D and 3-D models. Taken together, these observations indicate that, for studying pollutant toxicity during longer exposure times, 3-D cultures in direct contact with the pollutant seem to offer a more suitable approach - they mimic the in vivo behaviour of cells more realistically and under strictly controlled conditions. Thus, in readiness for possible forthcoming European regulations, we believe that the proposed study, even in its preliminary phase, can provide new advice on the assessment of the toxic and biological potential of particular chemical compounds derived from waste management processes. PMID:24512233

  11. Use of a twisted 3D Cauchy condition surface to reconstruct the last closed magnetic surface in a non-axisymmetric fusion plasma

    NASA Astrophysics Data System (ADS)

    Itagaki, Masafumi; Okubo, Gaku; Akazawa, Masayuki; Matsumoto, Yutaka; Watanabe, Kiyomasa; Seki, Ryosuke; Suzuki, Yasuhiro

    2012-12-01

    The three-dimensional (3D) Cauchy condition surface (CCS) method code, ‘CCS3D’, is now under development to reconstruct the 3D magnetic field profile outside a non-axisymmetric fusion plasma using only magnetic sensor signals. A new ‘twisted CCS’ is introduced, whose elliptic cross-section rotates with the variation in plasma geometry in the toroidal direction of a helical-type device. Independent of the toroidal angle, this CCS can be placed at a certain distance from the last closed magnetic surface (LCMS). With this new CCS, it is found through test calculations for the Large Helical Device that the numerical accuracy in the reconstructed field is improved. Furthermore, the magnetic field line tracing indicates the LCMS more precisely than with the use of the axisymmetric CCS. A new idea to determine the LCMS numerically is also proposed.

  12. Lentiviral shRNA knockdown of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture

    PubMed Central

    Coughlan, Teresa C; Crawford, Aileen; Goldring, Mary B; Hatton, Paul V; Barker, Michael D

    2010-01-01

    Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the 6 aggrecanases (ADAMTS-1, -4, -5, -8, -9 and -15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that 4 of the 6 aggrecanases are expressed in immortalized chondrocyte cell-lines and can be up-regulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knockdown of ADAMTS-5 and -9 expression in these cells, and by culturing them on 3 dimensional scaffolds, show that reduction in expression of ADAMTS-5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue-engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS-5 may be a therapeutic target in the treatment of arthritic disease. PMID:20568084

  13. Tailored and biodegradable poly(2-oxazoline) microbeads as 3D matrices for stem cell culture in regenerative therapies.

    PubMed

    Lück, Steffen; Schubel, René; Rüb, Jannick; Hahn, Dominik; Mathieu, Evelien; Zimmermann, Heike; Scharnweber, Dieter; Werner, Carsten; Pautot, Sophie; Jordan, Rainer

    2016-02-01

    We present the synthesis of hydrogel microbeads based on telechelic poly(2-oxazoline) (POx) crosslinkers and the methacrylate monomers (HEMA, METAC, SPMA) by inverse emulsion polymerization. While in batch experiments only irregular and ill-defined beads were obtained, the preparation in a microfluidic (MF) device resulted in highly defined hydrogel microbeads. Variation of the MF parameters allowed to control the microbead diameter from 50 to 500 μm. Microbead elasticity could be tuned from 2 to 20 kPa by the POx:monomer composition, the POx chain length, net charge of the hydrogel introduced via the monomer as well as by the organic content of the aqueous phase. The proliferations of human mesenchymal stem cells (hMSCs) on the microbeads were studied. While neutral, hydrophilic POx-PHEMA beads were bioinert, excessive colonization of hMSCs on charged POx-PMETAC and POx-PSPMA was observed. The number of proliferated cells scaled roughly linear with the METAC or SPMA comonomer content. Additional collagen I coating further improved the stem cell proliferation. Finally, a first POx-based system for the preparation of biodegradable hydrogel microcarriers is described and evaluated for stem cell culturing. PMID:26686977

  14. PPO/PEO modified hollow fiber membranes improved sensitivity of 3D cultured hepatocytes to drug toxicity via suppressing drug adsorption on membranes.

    PubMed

    Shen, Chong; Meng, Qin; He, Wenjuan; Wang, Qichen; Zhang, Guoliang

    2014-11-01

    The three dimensional (3D) cell culture in polymer-based micro system has become a useful tool for in vitro drug discovery. Among those polymers, polysulfone hollow fiber membrane (PSf HFM) is commonly used to create a microenvironment for cells. However, the target drug may adsorb on the polymeric surface, and this elicits negative impacts on cell exposure due to the reduced effective drug concentration in culture medium. In order to reduce the drug adsorption, PSf membrane were modified with hydrophilic Pluronic (PEO-b-PPO-b-PEO) copolymers, L121, P123 and F127 (PEO contents increase from 10%, 30% to 70%), by physical adsorption. As a result, the hydrophilicity of HFMs increased at an order of PSfF127>P123>L121 HFMs. The three modified membrane all showed significant resistance to adsorption of acid/neutral drugs. More importantly, the adsorption of base drugs were largely reduced to an average value of 11% on the L121 HFM. The improved resistance to drug adsorption could be attributed to the synergy of hydrophobic/neutrally charged PPO and hydrophilic PEO. The L121 HFM was further assessed by evaluating the drug hepatotoxicity in 3D culture of hepatocytes. The base drugs, clozapine and doxorubicin, showed more sensitive hepatotoxicity on hepatocytes in L121 HFM than in PSf HFM, while the acid drug, salicylic acid, showed the similar hepatotoxicity to hepatocytes in both HFMs. Our finding suggests that PSf HFM modified by PEO-b-PPO-b-PEO copolymers can efficiently resist the drug adsorption onto polymer membrane, and consequently improve the accuracy and sensitivity of in vitro hepatotoxic drug screening. PMID:25454662

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

    PubMed Central

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

    2014-01-01

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

  16. Novel method to dynamically load cells in 3D-gel culture for primary blast injury studies

    NASA Astrophysics Data System (ADS)

    Sory, David; Cepa-Areias, Anabela; Overby, Darryl; Proud, William; Institute of Shock Physics, Department of Bioengineering; Royal British Legion CentreBlast I Collaboration

    2015-06-01

    For at least a century explosive devices have been reported as one of the most important causes of injuries on battlefield in military conflicts as well as in terrorist attacks. Although significant experimental and modelling efforts have been focussed on blast injury at the organ or tissue level, few studies have investigated the mechanism of blast injury at the cellular level. This paper introduces an in vitro method compatible with living cells to examine the effects of high stress and short-duration pulses similar to those observed in blast waves. The experimental phase involved high strain rate axial compression of biological cylindrical specimens within a hermetically sealed sample holder made of a biocompatible polymer. Numerical simulations were performed in order to characterize the loading path within the sample and assess the loading conditions. A proof of concept is presented so as to establish a new window to address fundamental questions regarding primary blast injury at the cellular level. The Institute of Shock Physics acknowledges the support of AWE, Aldermaston, UK and Imperial College London. The Centre for Blast Injury Studies acknowledges the support of the Royal British Legion and Imperial College London.

  17. Naturally Derived Iron Oxide Nanowires from Bacteria for Magnetically Triggered Drug Release and Cancer Hyperthermia in 2D and 3D Culture Environments: Bacteria Biofilm to Potent Cancer Therapeutic.

    PubMed

    Kumeria, Tushar; Maher, Shaheer; Wang, Ye; Kaur, Gagandeep; Wang, Luoshan; Erkelens, Mason; Forward, Peter; Lambert, Martin F; Evdokiou, Andreas; Losic, Dusan

    2016-08-01

    Iron oxide nanowires produced by bacteria (Mariprofundus ferrooxydans) are demonstrated as new multifunctional drug carriers for triggered therapeutics release and cancer hyperthmia applications. Iron oxide nanowires are obtained from biofilm waste in the bore system used to pump saline groundwater into the River Murray, South Australia (Australia) and processed into individual nanowires with extensive magnetic properties. The drug carrier capabilities of these iron oxide nanowires (Bac-FeOxNWs) are assessed by loading anticancer drug (doxorubicin, Dox) followed by measuring its elution under sustained and triggered release conditions using alternating magnetic field (AMF). The cytotoxicity of Bac-FeOxNWs assessed in 2D (96 well plate) and 3D (Matrigel) cell cultures using MDA-MB231-TXSA human breast cancer cells and mouse RAW 264.7 macrophage cells shows that these Bac-FeOxNWs are biocompatible even at concentrations as high as 250 μg/mL after 24 h of incubation. Finally, we demonstrate the capabilities of Bac-FeOxNWs as potential hyperthermia agent in 3D culture setup. Application of AMF increased the local temperature by 14 °C resulting in approximately 34% decrease in cell viability. Our results demonstrate that these naturally produced nanowires in the form of biofilm can efficiently act as drug carriers with triggered payload release and magnetothermal heating features for potential anticancer therapeutics applications. PMID:27428076

  18. Combination of 12-O-tetradecanoylphorbol-13-acetate with diethyldithiocarbamate markedly inhibits pancreatic cancer cell growth in 3D culture and in immunodeficient mice

    PubMed Central

    HUANG, HUARONG; CAO, KAJIA; MALIK, SAQUIB; ZHANG, QIUYAN; LI, DONGLI; CHANG, RICHARD; WANG, HUAQIAN; LIN, WEIPING; VAN DOREN, JEREMIAH; ZHANG, KUN; DU, ZHIYUN; ZHENG, XI

    2015-01-01

    The aim of the present study was to determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC) alone or in combination on human pancreatic cancer cells cultured in vitro and grown as xenograft tumors in nude mice. Pancreatic cancer cells were treated with either DDTC or TPA alone, or in combination and the number of viable cells was then determined by trypan blue ecxlusion assay and the number of apoptotic cells was determined by morphological assessment by staining the cells with propidium idiode and examining them under a fluorescence microscope. Treatment with DDTC or TPA alone inhibited the growth and promoted the apoptosis of pancreatic cancer cells in a concentration-dependent manner. These effects were more prominent following treatment with TPA in combination with DDTC than following treatment with either agent alone in PANC-1 cells in monolayer cultures and in 3 dimensional (3D) cultures. The potent effects of the combination treatment on PANC-1 cells were associated with the inhibition of nuclear factor-κB (NF-κB) activation and the decreased expression of Bcl-2 induced by DDTC, as shown by NF-κB-dependent reporter gene expression assay and western blot analysis. Furthermore, treatment of nude mice with DDTC + TPA strongly inhibited the growth of PANC-1 xenograft tumors. The results of the present study indicate that the administration of TPA and DDTC in combination may be an effective strategy for inhibiting the growth of pancreatic cancer. PMID:25847449

  19. Responsiveness to PI3K and MEK Inhibitors in Breast Cancer. Use of a 3D Culture System to Study Pathways Related to Hormone Independence in Mice

    PubMed Central

    Polo, Maria Laura; Arnoni, Maria Victoria; Riggio, Marina; Wargon, Victoria; Lanari, Claudia; Novaro, Virginia

    2010-01-01

    Background A significant proportion of breast cancer patients face failure of endocrine therapy due to the acquisition of endocrine resistance. We have explored mechanisms involved in such disease progression by using a mouse breast cancer model that is induced by medroxyprogesterone acetate (MPA). These tumors transit through different stages of hormone sensitivity. However, when cells from tumor variants were seeded on plastic, all were stimulated by progestins and inhibited by antiprogestins such as RU486. Furthermore, cells from a RU486-resistant tumor variant recovered antiprogestin sensitivity. Hypothesis A three-dimensional (3D) culture system, by maintaining differential cellular organization that is typical of each tumor variant, may allow for the maintenance of particular hormone responses and thus be appropriate for the study of the effects of specific inhibitors of signaling pathways associated with disease progression. Method We compared the behavior of tumors growing in vivo and cancer cells ex vivo (in 3D Matrigel). In this system, we evaluated the effects of kinase inhibitors and hormone antagonists on tumor growth. Principal Findings LY294002, a PI3K/AKT pathway inhibitor, decreased both tumor growth in vivo and cell survival in Matrigel in MPA-independent tumors with higher AKT activity. Induction of cell death by anti-hormones such as ICI182780 and ZK230211 was more effective in MPA-dependent tumors with lower AKT activity. Inhibition of MEK with PD98059 did not affect tumor growth in any tested variant. Finally, while Matrigel reproduced differential responsiveness of MPA-dependent and -independent breast cancer cells, it was not sufficient to preserve antiprogestin resistance of RU486-resistant tumors. Conclusion We demonstrated that the PI3K/AKT pathway is relevant for MPA-independent tumor growth. Three-dimensional cultures were useful to test the effects of kinase inhibitors on breast cancer growth and highlight the need for in vivo models

  20. Combining Experimental Petrology and 3D Imaging to Gain Insight into Syn-eruptive Conditions of the Bishop Tuff, California

    NASA Astrophysics Data System (ADS)

    Chattin, Archer; Pamukcu, Ayla; Gardner, James; Gualda, Guilherme

    2015-04-01

    The Bishop tuff is a rhyolitic ignimbrite deposited by a supereruption 0.76 million years ago that formed the Long Valley Caldera in California, USA. Pamukcu et. al (2012) identifies two distinct crystal populations present in the Bishop Tuff, the first being a long-lived, large phenocryst population that records storage conditions, and the second a rapidly nucleated, quickly staunched microlite population thought to result from eruptive decompression. Laboratory experiments to reproduce this quickly grown population may help constrain the conditions and rates under which decompression took place. Rapid nucleation of microlites is accompanied by just as rapid bubble nucleation when volatiles exsolve during decompression; the size distribution of vesicles in eruptive products may thus provide important information on syn-eruptive processes. In this study we combine information from vesicle size distributions on natural pumice with data on experimentally produced microlite crystals with the goal of better understanding the syn-eruptive evolution of a supereruption-forming magma body. Decompression experiments are run using a natural Bishop tuff pumice clast ground and melted in the presence of water to obtain a melt representative of late-erupted Bishop Tuff (LBT) magmas. Experimental charges were subjected to decompression at varying rates and initial temperatures. At this time five experiments have been completed. All decompression experiments start at 130MPa, consistent with water concentration in LBT glass inclusions, and end at 10 MPa. Initial temperatures are either 710°C or 785°C, while decompression rates are 20 MPa/hr, 5.5MPa/hr, or 1.7MPa/hr Experimental products were compared to natural products using Scanning Electron Microscopy to document eventual crystal rims and microlites. We have been successful in causing limited feldspar crystallization, but have yet to generate quartz microlites. Bubble size distributions are obtained by analyzing x

  1. System for measuring oxygen consumption rates of mammalian cells in static culture under hypoxic conditions.

    PubMed

    Kagawa, Yuki; Miyahara, Hirotaka; Ota, Yuri; Tsuneda, Satoshi

    2016-01-01

    Estimating the oxygen consumption rates (OCRs) of mammalian cells in hypoxic environments is essential for designing and developing a three-dimensional (3-D) cell culture system. However, OCR measurements under hypoxic conditions are infrequently reported in the literature. Here, we developed a system for measuring OCRs at low oxygen levels. The system injects nitrogen gas into the environment and measures the oxygen concentration by an optical oxygen microsensor that consumes no oxygen. The developed system was applied to HepG2 cells in static culture. Specifically, we measured the spatial profiles of the local dissolved oxygen concentration in the medium, then estimated the OCRs of the cells. The OCRs, and also the pericellular oxygen concentrations, decreased nonlinearly as the oxygen partial pressure in the environment decreased from 19% to 1%. The OCRs also depended on the culture period and the matrix used for coating the dish surface. Using this system, we can precisely estimate the OCRs of various cell types under environments that mimic 3-D culture conditions, contributing crucial data for an efficient 3-D culture system design. PMID:26558344

  2. Automatic generation of boundary conditions using Demons non-rigid image registration for use in 3D modality-independent elastography

    NASA Astrophysics Data System (ADS)

    Pheiffer, Thomas S.; Ou, Jao J.; Miga, Michael I.

    2010-02-01

    Modality-independent elastography (MIE) is a method of elastography that reconstructs the elastic properties of tissue using images acquired under different loading conditions and a biomechanical model. Boundary conditions are a critical input to the algorithm, and are often determined by time-consuming point correspondence methods requiring manual user input. Unfortunately, generation of accurate boundary conditions for the biomechanical model is often difficult due to the challenge of accurately matching points between the source and target surfaces and consequently necessitates the use of large numbers of fiducial markers. This study presents a novel method of automatically generating boundary conditions by non-rigidly registering two image sets with a Demons diffusion-based registration algorithm. The use of this method was successfully performed in silico using magnetic resonance and X-ray computed tomography image data with known boundary conditions. These preliminary results have produced boundary conditions with accuracy of up to 80% compared to the known conditions. Finally, these boundary conditions were utilized within a 3D MIE reconstruction to determine an elasticity contrast ratio between tumor and normal tissue. Preliminary results show a reasonable characterization of the material properties on this first attempt and a significant improvement in the automation level and viability of the method.

  3. Experiments with osteoblasts cultured under hypergravity conditions

    NASA Technical Reports Server (NTRS)

    Kacena, Melissa A.; Todd, Paul; Gerstenfeld, Louis C.; Landis, William J.

    2004-01-01

    To understand further the role of gravity in osteoblast attachment, osteoblasts were subjected to hypergravity conditions in vitro. Scanning electron microscopy of all confluent coverslips from FPA units show that the number of attached osteoblasts was similar among gravitational levels and growth durations (90 cells/microscopic field). Specifically, confluent 1.0 G control cultures contained an average of 91 +/- 8 cells/field, 3.3 G samples had 88 +/- 8 cells/field, and 4.0 G cultures averaged 90 +/- 7 cells/field. The sparsely plated cultures assessed by immunohistochemistry also had similar numbers of cells at each time point (l.0 G was similar to 3.3 and 4.0 G), but cell number changed from one time point to the next as those cells proliferated. Immunohistochemistry of centrifuged samples showed an increase in number (up to 160% increase) and thickness (up to 49% increase) of actin fibers, a decrease in intensity of fibronectin fluorescence (18-23% decrease) and an increase in number of vinculin bulbs (202-374% increase in number of vinculin bulbs/area). While hypergravity exposure did not alter the number of attached osteoblasts, it did result in altered actin, fibronectin, and vinculin elements, changing some aspects of osteoblast- substrate adhesion.

  4. Probing tumor-stroma interactions and response to photodynamic therapy in a 3D pancreatic cancer-fibroblast co-culture model

    NASA Astrophysics Data System (ADS)

    Glidden, Michael D.; Massodi, Iqbal; Rizvi, Imran; Celli, Jonathan P.; Hasan, Tayyaba

    2012-02-01

    Pancreatic ductal adenocarcinoma is a lethal disease that is often unresectable by the time of diagnosis and is typically non-responsive to chemo- and radiotherapy, resulting in a five year survival of only 3%. Tumors of the pancreas are characterized by a dense fibrous stroma rich in extracellular matrix proteins, which is implicated in poor therapeutic response, though its precise roles remain poorly understood. Indeed, while the use of therapeutics that target the stroma is an emerging paradigm in the clinical management of this disease, the primary focus of such efforts is to enhance drug penetration through dense fibrous stroma and it is unclear to what extent the characteristically rigid stroma of pancreatic tumors imparts drug resistance by acting as a complex signaling partner, or merely as a physical barrier for drug delivery. Here we use 3D in vitro co-cultures of pancreatic cancer cells and normal human fibroblasts as a model system to study heterotypic interactions between these populations. Leveraging this in vitro model along with image-based methods for quantification of growth and therapeutic endpoints, we characterize these co-cultures and examine the role of verteporfin-based photodynamic therapy (PDT) for targeting tumor-fibroblast interactions in pancreatic tumors.

  5. Effect of ionizing radiation on acinar morphogenesis of human prostatic epithelial cells under three-dimensional culture conditions.

    PubMed

    Wang, T; X, S Ma; Kong, D; Yi, H; Wang, X; Liang, B; Xu, H; He, M; Jia, L; Qased, A B; Yang, Y; Liu, X

    2012-01-01

    Homeostasis is maintained by the interplay of multiple factors that directly or indirectly regulate cell proliferation and cell death. Complex multiple interactions between cells and the extracellular matrix occur during acinar morphogenesis and changes in these might indicate carcinogenesis of cells from a normal to a malignant, invasive phenotype. In this study, the human prostatic epithelial cell line RWPE-1 was cultured under three-dimensional (3-D) culture conditions, and the effect of ionizing radiation on acinar morphogenesis and its association with autophagy were discussed. The results illustrated that formation of specific spheroid (acinar) structures was detectable under 3-D culture conditions. Radiation induced the disruption of acini in different cell models using either gene overexpression (Akt) or gene knock-down (Beclin 1 and ATG7). Introduction of Akt not only accelerated the growth of cells (i.e., caused the cells to manifest elongating and microspike-like structures that are obviously different from structures seen in wild-type RWPE-1 cells under two-dimensional conditions), but also changed their morphological characteristics under 3-D culture conditions. Knock-down of autophagy-related genes (Beclin 1 and ATG7) increased the radiosensitivity of cells under 3-D culture conditions, and cells died of non-apoptotic death after radiation. The results suggested that ionizing radiation may change the cell phenotype and the formation of acini. Additionally even the autophagy mechanism may play a role in these processes. PMID:22296497

  6. Self-Supported Cedarlike Semimetallic Cu3P Nanoarrays as a 3D High-Performance Janus Electrode for Both Oxygen and Hydrogen Evolution under Basic Conditions.

    PubMed

    Hou, Chun-Chao; Chen, Qian-Qian; Wang, Chuan-Jun; Liang, Fei; Lin, Zheshuai; Fu, Wen-Fu; Chen, Yong

    2016-09-01

    There has been strong and growing interest in the development of cost-effective and highly active oxygen evolution reaction (OER) electrocatalysts for alternative fuels utilization and conversion devices. We report herein that semimetallic Cu3P nanoarrays directly grown on 3D copper foam (CF) substrate can function as effective electrocatalysts for water oxidation. Specifically, the surface oxidation-activated Cu3P only required a relatively low overpotential of 412 mV to achieve a current density of 50 mA cm(-2) and displayed a small Tafel slope of 63 mV dec(-1) in 0.1 M KOH solution, on account of the collaborative effect of large roughness factor (RF) and semimetallic character. Following that, investigations into the mechanism revealed the formation of a unique active phase during the water oxidation process in which conductive Cu3P was the core covered with a thin copper oxide/hydroxide layer. Moreover, this Cu3P 3D electrode was also applied to the hydrogen evolution reaction (HER) and showed good catalytic performance and stability under the same basic conditions. PMID:27559613

  7. Final predictions of ambient conditions along the east-west crossdrift using the 3-D UZ site-scale model. Level 4 milestoneSP33ABM4.

    SciTech Connect

    Ritcey, A.C.; Sonnenthal, E.L.; Wu, Y.S.; Haukwa, C.; Bodvarsson,G.S.

    1998-03-01

    In 1998, the Yucca Mountain Site Characterization Project (YMP) is expected to continue construction of an East-West Cross Drift. The 5-meter diameter drift will extend from the North Ramp of the Exploratory Studies Facility (ESF), near Station 19+92, southwest through the repository block, and over to and through the Solitario Canyon Fault. This drift is part of a program designed to enhance characterization of Yucca Mountain and to complement existing surface-based and ESF testing studies. The objective of this milestone is to use the three-dimensional (3-D) unsaturated zone (UZ) site-scale model to predict ambient conditions along the East-West Cross Drift. These predictions provide scientists and engineers with a priori information that can support design and construction of the East-West Cross Drift and associated testing program. The predictions also provide, when compared with data collected after drift construction, an opportunity to test and verify the calibration of the 3-D UZ site-scale model.

  8. Europeana and 3D

    NASA Astrophysics Data System (ADS)

    Pletinckx, D.

    2011-09-01

    The current 3D hype creates a lot of interest in 3D. People go to 3D movies, but are we ready to use 3D in our homes, in our offices, in our communication? Are we ready to deliver real 3D to a general public and use interactive 3D in a meaningful way to enjoy, learn, communicate? The CARARE project is realising this for the moment in the domain of monuments and archaeology, so that real 3D of archaeological sites and European monuments will be available to the general public by 2012. There are several aspects to this endeavour. First of all is the technical aspect of flawlessly delivering 3D content over all platforms and operating systems, without installing software. We have currently a working solution in PDF, but HTML5 will probably be the future. Secondly, there is still little knowledge on how to create 3D learning objects, 3D tourist information or 3D scholarly communication. We are still in a prototype phase when it comes to integrate 3D objects in physical or virtual museums. Nevertheless, Europeana has a tremendous potential as a multi-facetted virtual museum. Finally, 3D has a large potential to act as a hub of information, linking to related 2D imagery, texts, video, sound. We describe how to create such rich, explorable 3D objects that can be used intuitively by the generic Europeana user and what metadata is needed to support the semantic linking.

  9. Real-time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor.

    PubMed

    Sriram, Renuka; Van Criekinge, Mark; Hansen, Ailin; Wang, Zhen J; Vigneron, Daniel B; Wilson, David M; Keshari, Kayvan R; Kurhanewicz, John

    2015-09-01

    We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) (13)C MR and interrogated HP [1-(13)C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP (13)C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra- (Lacin ) and extracellular (Lacex ) HP lactate signals were robustly resolved in dynamic (13)C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1-(13)C]pyruvate delivery, the ratio of HP Lacin /Lacex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p < 0.05) increase in the Lacex pool size. Lacin /Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient-derived RCC tissue slices using HP [1,2-(13)C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5-(13)C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export. PMID

  10. Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D.

    PubMed

    Kleinhans, C; Schmid, F F; Schmid, F V; Kluger, P J

    2015-07-10

    Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVβ3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts. PMID:25562421

  11. Differential osteogenicity of multiple donor-derived human mesenchymal stem cells and osteoblasts in monolayer, scaffold-based 3D culture and in vivo.

    PubMed

    Quent, Verena M C; Theodoropoulos, Christina; Hutmacher, Dietmar W; Reichert, Johannes C

    2016-06-01

    We set out to compare the osteogenicity of human mesenchymal stem (hMSCs) and osteoblasts (hOBs). Upon osteogenic induction in monolayer, hMSCs showed superior matrix mineralization expressing characteristic bone-related genes. For scaffold cultures, both cell types presented spindle-shaped, osteoblast-like morphologies forming a dense, interconnected network of high viability. On the scaffolds, hOBs proliferated faster. A general upregulation of parathyroid hormone-related protein (PTHrP), osteoprotegrin (OPG), receptor activator of NF-κB ligand (RANKL), sclerostin (SOST), and dentin matrix protein 1 (DMP1) was observed for both cell types. Simultaneously, PTHrP, RANKL and DMP-1 expression decreased under osteogenic stimulation, while OPG and SOST increased significantly. Following transplantation into NOD/SCID mice, μCT and histology showed increased bone deposition with hOBs. The bone was vascularized, and amounts further increased for both cell types after recombinant human bone morphogenic protein 7 (rhBMP-7) addition also stimulating osteoclastogenesis. Complete bone organogenesis was evidenced by the presence of osteocytes and hematopoietic precursors. Our study results support the asking to develop 3D cellular models closely mimicking the functions of living tissues suitable for in vivo translation. PMID:25781662

  12. Fdf in US3D

    NASA Astrophysics Data System (ADS)

    Otis, Collin; Ferrero, Pietro; Candler, Graham; Givi, Peyman

    2013-11-01

    The scalar filtered mass density function (SFMDF) methodology is implemented into the computer code US3D. This is an unstructured Eulerian finite volume hydrodynamic solver and has proven very effective for simulation of compressible turbulent flows. The resulting SFMDF-US3D code is employed for large eddy simulation (LES) on unstructured meshes. Simulations are conducted of subsonic and supersonic flows under non-reacting and reacting conditions. The consistency and the accuracy of the simulated results are assessed along with appraisal of the overall performance of the methodology. The SFMDF-US3D is now capable of simulating high speed flows in complex configurations.

  13. 3d-3d correspondence revisited

    NASA Astrophysics Data System (ADS)

    Chung, Hee-Joong; Dimofte, Tudor; Gukov, Sergei; Sułkowski, Piotr

    2016-04-01

    In fivebrane compactifications on 3-manifolds, we point out the importance of all flat connections in the proper definition of the effective 3d {N}=2 theory. The Lagrangians of some theories with the desired properties can be constructed with the help of homological knot invariants that categorify colored Jones polynomials. Higgsing the full 3d theories constructed this way recovers theories found previously by Dimofte-Gaiotto-Gukov. We also consider the cutting and gluing of 3-manifolds along smooth boundaries and the role played by all flat connections in this operation.

  14. Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates

    PubMed Central

    Amer, Luke D.; Holtzinger, Audrey; Keller, Gordon; Mahoney, Melissa J.; Bryant, Stephanie J.

    2015-01-01

    This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15) μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced in both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (~2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1+/Nkx6.1+ cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates. PMID:25913222

  15. 3D acoustic wave modelling with time-space domain dispersion-relation-based finite-difference schemes and hybrid absorbing boundary conditions

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Sen, Mrinal K.

    2011-09-01

    Most conventional finite-difference methods adopt second-order temporal and (2M)th-order spatial finite-difference stencils to solve the 3D acoustic wave equation. When spatial finite-difference stencils devised from the time-space domain dispersion relation are used to replace these conventional spatial finite-difference stencils devised from the space domain dispersion relation, the accuracy of modelling can be increased from second-order along any directions to (2M)th-order along 48 directions. In addition, the conventional high-order spatial finite-difference modelling accuracy can be improved by using a truncated finite-difference scheme. In this paper, we combine the time-space domain dispersion-relation-based finite difference scheme and the truncated finite-difference scheme to obtain optimised spatial finite-difference coefficients and thus to significantly improve the modelling accuracy without increasing computational cost, compared with the conventional space domain dispersion-relation-based finite difference scheme. We developed absorbing boundary conditions for the 3D acoustic wave equation, based on predicting wavefield values in a transition area by weighing wavefield values from wave equations and one-way wave equations. Dispersion analyses demonstrate that high-order spatial finite-difference stencils have greater accuracy than low-order spatial finite-difference stencils for high frequency components of wavefields, and spatial finite-difference stencils devised in the time-space domain have greater precision than those devised in the space domain under the same discretisation. The modelling accuracy can be improved further by using the truncated spatial finite-difference stencils. Stability analyses show that spatial finite-difference stencils devised in the time-space domain have better stability condition. Numerical modelling experiments for homogeneous, horizontally layered and Society of Exploration Geophysicists/European Association of

  16. Imaging a Sustainable Future in 3D

    NASA Astrophysics Data System (ADS)

    Schuhr, W.; Lee, J. D.; Kanngieser, E.

    2012-07-01

    It is the intention of this paper, to contribute to a sustainable future by providing objective object information based on 3D photography as well as promoting 3D photography not only for scientists, but also for amateurs. Due to the presentation of this article by CIPA Task Group 3 on "3D Photographs in Cultural Heritage", the presented samples are masterpieces of historic as well as of current 3D photography concentrating on cultural heritage. In addition to a report on exemplarily access to international archives of 3D photographs, samples for new 3D photographs taken with modern 3D cameras, as well as by means of a ground based high resolution XLITE staff camera and also 3D photographs taken from a captive balloon and the use of civil drone platforms are dealt with. To advise on optimum suited 3D methodology, as well as to catch new trends in 3D, an updated synoptic overview of the 3D visualization technology, even claiming completeness, has been carried out as a result of a systematic survey. In this respect, e.g., today's lasered crystals might be "early bird" products in 3D, which, due to lack in resolution, contrast and color, remember to the stage of the invention of photography.

  17. 3D and Education

    NASA Astrophysics Data System (ADS)

    Meulien Ohlmann, Odile

    2013-02-01

    Today the industry offers a chain of 3D products. Learning to "read" and to "create in 3D" becomes an issue of education of primary importance. 25 years professional experience in France, the United States and Germany, Odile Meulien set up a personal method of initiation to 3D creation that entails the spatial/temporal experience of the holographic visual. She will present some different tools and techniques used for this learning, their advantages and disadvantages, programs and issues of educational policies, constraints and expectations related to the development of new techniques for 3D imaging. Although the creation of display holograms is very much reduced compared to the creation of the 90ies, the holographic concept is spreading in all scientific, social, and artistic activities of our present time. She will also raise many questions: What means 3D? Is it communication? Is it perception? How the seeing and none seeing is interferes? What else has to be taken in consideration to communicate in 3D? How to handle the non visible relations of moving objects with subjects? Does this transform our model of exchange with others? What kind of interaction this has with our everyday life? Then come more practical questions: How to learn creating 3D visualization, to learn 3D grammar, 3D language, 3D thinking? What for? At what level? In which matter? for whom?

  18. Adipose-derived mesenchymal stem cells from the sand rat: transforming growth factor beta and 3D co-culture with human disc cells stimulate proteoglycan and collagen type I rich extracellular matrix

    PubMed Central

    Tapp, Hazel; Deepe, Ray; Ingram, Jane A; Kuremsky, Marshall; Hanley, Edward N; Gruber, Helen E

    2008-01-01

    Introduction Adult mesenchymal stem cell therapy has a potential application in the biological treatment of disc degeneration. Our objectives were: to direct adipose-derived mesenchymal stem cells (AD-MSC) from the sand rat to produce a proteoglycan and collagen type I extracellular matrix (ECM) rich in known ECM components of the annulus fibrosis of disc; and to stimulate proteoglycan production by co-culture of human annulus cells with AD-MSC. Methods AD-MSC were isolated and characterised by adherence to plastic, appropriate expression of cluster of differentiation (CD) markers, and differentiation to osteoblasts and chondrocytes in vitro. AD-MSC were grown in three-dimensional (3D) culture and treated with or without transforming growth factor beta (TGFβ) to direct them to produce annulus-like ECM as determined by proteoglycan content and collagen expression. AD-MSC were co-cultured with human annulus cells and grown in 3D culture. Results AD-MSC produced a proteoglycan and collagen type I rich ECM after treatment with TGFβ in 3D culture as confirmed by a 48% increase in proteoglycan content assayed by 1,9-dimethylmethylene blue (DMB), and by immunohistochemical identification of ECM components. Co-culture of human annulus and sand rat AD-MSC in 3D culture resulted in a 20% increase in proteoglycan production compared with the predicted value of the sum of the individual cultures. Conclusion Results support the hypothesis that AD-MSC have potential in cell-based therapy for disc degeneration. PMID:18691412

  19. Conditioning Factors of an Organizational Learning Culture

    ERIC Educational Resources Information Center

    Rebelo, Teresa Manuela; Gomes, Adelino Duarte

    2011-01-01

    Purpose: The aim of this study is to assess the relationship between some variables (organizational structure, organizational dimension and age, human resource characteristics, the external environment, strategy and quality) and organizational learning culture and evaluate the way they interact with this kind of culture.…

  20. 3D Imaging.

    ERIC Educational Resources Information Center

    Hastings, S. K.

    2002-01-01

    Discusses 3 D imaging as it relates to digital representations in virtual library collections. Highlights include X-ray computed tomography (X-ray CT); the National Science Foundation (NSF) Digital Library Initiatives; output peripherals; image retrieval systems, including metadata; and applications of 3 D imaging for libraries and museums. (LRW)

  1. Using Global Plate Velocity Boundary Conditions for Embedded Regional Geodynamic Models: Application to 3-D Modeling of the Early Rifting of the South Atlantic

    NASA Astrophysics Data System (ADS)

    Taramón, Jorge M.; Morgan, Jason P.; Pérez-Gussinyé, Marta

    2016-04-01

    The treatment of far-field boundary conditions is one of the most poorly resolved issues for regional modeling of geodynamic processes. In viscous flow, the choice of far-field boundary conditions often strongly shapes the large-scale structure of a geosimulation. The mantle velocity field along the sidewalls and base of a modeling region is typically much more poorly known than the geometry of past global motions of the surface plates as constrained by global plate motion reconstructions. For regional rifting models it has become routine to apply highly simplified 'plate spreading' or 'uniform rifting' boundary conditions to a 3-D model that limits its ability to simulate the geodynamic evolution of a specific rifted margin. One way researchers are exploring the sensitivity of regional models to uncertain boundary conditions is to use a nested modeling approach in which a global model is used to determine a large-scale flow pattern that is imposed as a constraint along the boundaries of the region to be modeled. Here we explore the utility of a different approach that takes advantage of the ability of finite element models to use unstructured meshes than can embed much higher resolution sub-regions. Here we demonstrate the workflow and code tools that we created to generate this unstructured mesh: solver based on springs, guide-mesh and routines to improve the quality, e.g., closeness to a regular tetrahedron, of the tetrahedral elements of the mesh. Note that the same routines are used to generate a new mesh in the remeshing of a distorted Lagrangian mesh. In our initial project to validate this approach, we create a global spherical shell mesh in which a higher resolution sub-region is created around the nascent South Atlantic Rifting Margin. Global Plate motion BCs and plate boundaries are applied for the time of the onset of rifting, continuing through several 10s of Ma of rifting. Thermal, compositional, and melt-related buoyancy forces are only non

  2. Simple and Versatile Turbidimetric Monitoring of Bacterial Growth in Liquid Cultures Using a Customized 3D Printed Culture Tube Holder and a Miniaturized Spectrophotometer: Application to Facultative and Strictly Anaerobic Bacteria

    PubMed Central

    Maia, Margarida R. G.; Marques, Sara; Cabrita, Ana R. J.; Wallace, R. John; Thompson, Gertrude; Fonseca, António J. M.; Oliveira, Hugo M.

    2016-01-01

    Here we introduce a novel strategy for turbidimetric monitoring of bacterial growth in liquid culture. The instrumentation comprises a light source, a customized 3D printed culture tube holder and a miniaturized spectrophotometer, connected through optical cables. Due to its small footprint and the possibility to operate with external light, bacterial growth was directly monitored from culture tubes in a simple and versatile fashion. This new portable measurement technique was used to monitor the growth of facultative (Escherichia coli ATCC/25922, and Staphylococcus aureus ATCC/29213) and strictly (Butyrivibrio fibrisolvens JW11, Butyrivibrio proteoclasticus P18, and Propionibacterium acnes DSMZ 1897) anaerobic bacteria. For E. coli and S. aureus, the growth rates calculated from normalized optical density values were compared with those ones obtained using a benchtop spectrophotometer without significant differences (P = 0.256). For the strictly anaerobic species, a high precision (relative standard deviation < 3.5%) was observed between replicates up to 48 h. Regarding its potential for customization, this manifold could accommodate further developments for customized turbidimetric monitoring, such as the use of light-emitting diodes as a light source or flow cells.

  3. Experiments performed with bubbly flow in vertical pipes at different flow conditions covering the transition region: simulation by coupling Eulerian, Lagrangian and 3D random walks models

    NASA Astrophysics Data System (ADS)

    Muñoz-Cobo, José; Chiva, Sergio; El Aziz Essa, Mohamed; Mendes, Santos

    2012-08-01

    Two phase flow experiments with different superficial velocities of gas and water were performed in a vertical upward isothermal cocurrent air-water flow column with conditions ranging from bubbly flow, with very low void fraction, to transition flow with some cap and slug bubbles and void fractions around 25%. The superficial velocities of the liquid and the gas phases were varied from 0.5 to 3 m/s and from 0 to 0.6 m/s, respectively. Also to check the effect of changing the surface tension on the previous experiments small amounts of 1-butanol were added to the water. These amounts range from 9 to 75 ppm and change the surface tension. This study is interesting because in real cases the surface tension of the water diminishes with temperature, and with this kind of experiments we can study indirectly the effect of changing the temperature on the void fraction distribution. The following axial and radial distributions were measured in all these experiments: void fraction, interfacial area concentration, interfacial velocity, Sauter mean diameter and turbulence intensity. The range of values of the gas superficial velocities in these experiments covered the range from bubbly flow to the transition to cap/slug flow. Also with transition flow conditions we distinguish two groups of bubbles in the experiments, the small spherical bubbles and the cap/slug bubbles. Special interest was devoted to the transition region from bubbly to cap/slug flow; the goal was to understand the physical phenomena that take place during this transition A set of numerical simulations of some of these experiments for bubbly flow conditions has been performed by coupling a Lagrangian code, that tracks the three dimensional motion of the individual bubbles in cylindrical coordinates inside the field of the carrier liquid, to an Eulerian model that computes the magnitudes of continuous phase and to a 3D random walk model that takes on account the fluctuation in the velocity field of the

  4. A study of the variation of physical conditions in the cometary coma based on a 3D multi-fluid model

    NASA Astrophysics Data System (ADS)

    Shou, Y.; Combi, M. R.; Fougere, N.; Tenishev, V.; Toth, G.; Gombosi, T. I.; Huang, Z.; Jia, X.; Bieler, A. M.; Hansen, K. C.

    2015-12-01

    Physics-based numerical coma models are desirable whether to interpret the spacecraft observations of the inner coma or to compare with the ground-based observations of the outer coma. One example is Direct Simulation Monte Carlo (DSMC) method, which has been successfully adopted to simulate the coma under various complex conditions. However, for bright comets with large production rates, the time step in DSMC model has to be tiny to accommodate the small mean free path and the high collision frequency. In addition a truly time-variable 3D DSMC model would still be computationally difficult or even impossible under most circumstances. In this work, we develop a multi-neutral-fluid model based on BATS-R-US in the University of Michigan's SWMF (Space Weather Modeling Framework), which can serve as a useful alternative to DSMC methods to compute both the inner and the outer coma and to treat time-variable phenomena. This model treats H2O, OH, H2, O, H and CO2 as separate fluids and each fluid has its own velocity and temperature. But collisional interactions can also couple all fluids together. Collisional interactions tend to decrease the velocity differences and are also able to re-distribute the excess energy deposited by chemical reactions among all species. To compute the momentum and energy transfer caused by such interactions self-consistently, collisions between fluids, whose efficiency is proportional to the densities, are included as well as heating from various chemical reactions. By applying the model to comets with different production rates (i.e. 67P/Churyumov-Gerasimenko, 1P/Halley, etc.), we are able to study how the heating efficiency varies with cometocentric distances and production rates. The preliminary results and comparison are presented and discussed. This work has been partially supported by grant NNX14AG84G from the NASA Planetary Atmospheres Program, and US Rosetta contracts JPL #1266313, JPL #1266314 and JPL #1286489.

  5. Changes in gene expression, protein content and morphology of chondrocytes cultured on a 3D Random Positioning Machine and 2D rotating clinostat

    NASA Astrophysics Data System (ADS)

    Aleshcheva, Ganna; Hauslage, Jens; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela; Sahana, Jayashree

    Chondrocytes are the only cell type found in human cartilage consisting of proteoglycans and type II collagen. Several studies on chondrocytes cultured either in Space or on a ground-based facility for simulation of microgravity revealed that these cells are very resistant to adverse effects and stress induced by altered gravity. Tissue engineering of chondrocytes is a new strategy for cartilage regeneration. Using a three-dimensional Random Positioning Machine and a 2D rotating clinostat, devices designed to simulate microgravity on Earth, we investigated the early effects of microgravity exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis; and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced for 24 h. Based on the results achieved, we suggest that chondrocytes exposed to simulated microgravity seem to change their extracellular matrix production behavior while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.

  6. TRACE 3-D documentation

    SciTech Connect

    Crandall, K.R.

    1987-08-01

    TRACE 3-D is an interactive beam-dynamics program that calculates the envelopes of a bunched beam, including linear space-charge forces, through a user-defined transport system. TRACE 3-D provides an immediate graphics display of the envelopes and the phase-space ellipses and allows nine types of beam-matching options. This report describes the beam-dynamics calculations and gives detailed instruction for using the code. Several examples are described in detail.

  7. Cross-Cultural Discussions in a 3D Virtual Environment and Their Affordances for Learners' Motivation and Foreign Language Discussion Skills

    ERIC Educational Resources Information Center

    Jauregi, Kristi; Kuure, Leena; Bastian, Pim; Reinhardt, Dennis; Koivisto, Tuomo

    2015-01-01

    Within the European TILA project a case study was carried out where pupils from schools in Finland and the Netherlands engaged in debating sessions using the 3D virtual world of OpenSim once a week for a period of 5 weeks. The case study had two main objectives: (1) to study the impact that the discussion tasks undertaken in a virtual environment…

  8. Image-Based and Range-Based 3d Modelling of Archaeological Cultural Heritage: the Telamon of the Temple of Olympian ZEUS in Agrigento (italy)

    NASA Astrophysics Data System (ADS)

    Lo Brutto, M.; Spera, M. G.

    2011-09-01

    The Temple of Olympian Zeus in Agrigento (Italy) was one of the largest temple and at the same time one of the most original of all the Greek architecture. We don't know exactly how it was because the temple is now almost completely destroyed but it is very well-known for the presence of the Telamons. The Telamons were giant statues (about 8 meters high) probably located outside the temple to fill the interval between the columns. In accordance with the theory most accredited by archaeologists the Telamons were a decorative element and also a support for the structure. However, this hypothesis has never been scientifically proven. One Telamon has been reassembled and is shown at the Archaeological Museum of Agrigento. In 2009 a group of researchers at the University of Palermo has begun a study to test the hypothesis that the Telamons support the weight of the upper part of the temple. The study consists of a 3D survey of the Telamon, to reconstruct a detailed 3D digital model, and of a structural analysis with the Finite Element Method (FEM) to test the possibility that the Telamon could to support the weight of the upper portion of the temple. In this work the authors describe the 3D survey of Telamon carry out with Range-Based Modelling (RBM) and Image-Based Modeling (IBM). The RBM was performed with a TOF laser scanner while the IBM with the ZScan system of Menci Software and Image Master of Topcon. Several tests were conducted to analyze the accuracy of the different 3D models and to evaluate the difference between laser scanning and photogrammetric data. Moreover, an appropriate data reduction to generate a 3D model suitable for FEM analysis was tested.

  9. Functional 3D human primary hepatocyte spheroids made by co-culturing hepatocytes from partial hepatectomy specimens and human adipose-derived stem cells.

    PubMed

    No, Da Yoon; Lee, Seung-A; Choi, Yoon Young; Park, DoYeun; Jang, Ju Yun; Kim, Dong-Sik; Lee, Sang-Hoon

    2012-01-01

    We have generated human hepatocyte spheroids with uniform size and shape by co-culturing 1∶1 mixtures of primary human hepatocytes (hHeps) from partial hepatectomy specimens and human adipose-derived stem cells (hADSCs) in concave microwells. The hADSCs in spheroids could compensate for the low viability and improve the functional maintenance of hHeps. Co-cultured spheroids aggregated and formed compact spheroidal shapes more rapidly, and with a significantly higher viability than mono-cultured spheroids. The liver-specific functions of co-cultured spheroids were greater, although they contained half the number of hepatocytes as mono-cultured spheroids. Albumin secretion by co-cultured spheroids was 10% higher on day 7, whereas urea secretion was similar, compared with mono-cultured spheroids. A quantitative cytochrome P450 assay showed that the enzymatic activity of co-cultured spheroids cultured for 9 days was 28% higher than that of mono-cultured spheroids. These effects may be due to the transdifferentiation potential and paracrine healing effects of hADSCs on hHeps. These co-cultured spheroids may be useful for creating artificial three-dimensional hepatic tissue constructs and for cell therapy with limited numbers of human hepatocytes. PMID:23236387

  10. Stiffness of the microenvironment upregulates ERBB2 expression in 3D cultures of MCF10A within the range of mammographic density.

    PubMed

    Cheng, Qingsu; Bilgin, Cemal Cagatay; Fonteney, Gerald; Chang, Hang; Henderson, Matthew; Han, Ju; Parvin, Bahram

    2016-01-01

    The effects of the stiffness of the microenvironment on the molecular response of 3D colony organization, at the maximum level of mammographic density (MD), are investigated. Phenotypic profiling reveals that 3D colony formation is heterogeneous and increased stiffness of the microenvironment, within the range of the MD, correlates with the increased frequency of aberrant 3D colony formation. Further integrative analysis of the genome-wide transcriptome and phenotypic profiling hypothesizes overexpression of ERBB2 in the premalignant MCF10A cell lines at a stiffness value that corresponds to the collagen component at high mammographic density. Subsequently, ERBB2 overexpression has been validated in the same cell line. Similar experiments with a more genetically stable cell line of 184A1 also revealed an increased frequency of aberrant colony formation with the increased stiffness; however, 184A1 did not demonstrate overexpression of ERBB2 at the same stiffness value of the high MD. These results suggest that stiffness exacerbates premalignant cell line of MCF10A. PMID:27383056

  11. Stiffness of the microenvironment upregulates ERBB2 expression in 3D cultures of MCF10A within the range of mammographic density

    PubMed Central

    Cheng, Qingsu; Bilgin, Cemal Cagatay; Fonteney, Gerald; Chang, Hang; Henderson, Matthew; Han, Ju; Parvin, Bahram

    2016-01-01

    The effects of the stiffness of the microenvironment on the molecular response of 3D colony organization, at the maximum level of mammographic density (MD), are investigated. Phenotypic profiling reveals that 3D colony formation is heterogeneous and increased stiffness of the microenvironment, within the range of the MD, correlates with the increased frequency of aberrant 3D colony formation. Further integrative analysis of the genome-wide transcriptome and phenotypic profiling hypothesizes overexpression of ERBB2 in the premalignant MCF10A cell lines at a stiffness value that corresponds to the collagen component at high mammographic density. Subsequently, ERBB2 overexpression has been validated in the same cell line. Similar experiments with a more genetically stable cell line of 184A1 also revealed an increased frequency of aberrant colony formation with the increased stiffness; however, 184A1 did not demonstrate overexpression of ERBB2 at the same stiffness value of the high MD. These results suggest that stiffness exacerbates premalignant cell line of MCF10A. PMID:27383056

  12. Radiochromic 3D Detectors

    NASA Astrophysics Data System (ADS)

    Oldham, Mark

    2015-01-01

    Radiochromic materials exhibit a colour change when exposed to ionising radiation. Radiochromic film has been used for clinical dosimetry for many years and increasingly so recently, as films of higher sensitivities have become available. The two principle advantages of radiochromic dosimetry include greater tissue equivalence (radiologically) and the lack of requirement for development of the colour change. In a radiochromic material, the colour change arises direct from ionising interactions affecting dye molecules, without requiring any latent chemical, optical or thermal development, with important implications for increased accuracy and convenience. It is only relatively recently however, that 3D radiochromic dosimetry has become possible. In this article we review recent developments and the current state-of-the-art of 3D radiochromic dosimetry, and the potential for a more comprehensive solution for the verification of complex radiation therapy treatments, and 3D dose measurement in general.

  13. Bootstrapping 3D fermions

    NASA Astrophysics Data System (ADS)

    Iliesiu, Luca; Kos, Filip; Poland, David; Pufu, Silviu S.; Simmons-Duffin, David; Yacoby, Ran

    2016-03-01

    We study the conformal bootstrap for a 4-point function of fermions < ψψψψ> in 3D. We first introduce an embedding formalism for 3D spinors and compute the conformal blocks appearing in fermion 4-point functions. Using these results, we find general bounds on the dimensions of operators appearing in the ψ × ψ OPE, and also on the central charge C T . We observe features in our bounds that coincide with scaling dimensions in the GrossNeveu models at large N . We also speculate that other features could coincide with a fermionic CFT containing no relevant scalar operators.

  14. TACO3D. 3-D Finite Element Heat Transfer Code

    SciTech Connect

    Mason, W.E.

    1992-03-04

    TACO3D is a three-dimensional, finite-element program for heat transfer analysis. An extension of the two-dimensional TACO program, it can perform linear and nonlinear analyses and can be used to solve either transient or steady-state problems. The program accepts time-dependent or temperature-dependent material properties, and materials may be isotropic or orthotropic. A variety of time-dependent and temperature-dependent boundary conditions and loadings are available including temperature, flux, convection, and radiation boundary conditions and internal heat generation. Additional specialized features treat enclosure radiation, bulk nodes, and master/slave internal surface conditions (e.g., contact resistance). Data input via a free-field format is provided. A user subprogram feature allows for any type of functional representation of any independent variable. A profile (bandwidth) minimization option is available. The code is limited to implicit time integration for transient solutions. TACO3D has no general mesh generation capability. Rows of evenly-spaced nodes and rows of sequential elements may be generated, but the program relies on separate mesh generators for complex zoning. TACO3D does not have the ability to calculate view factors internally. Graphical representation of data in the form of time history and spatial plots is provided through links to the POSTACO and GRAPE postprocessor codes.

  15. Tooteko: a Case Study of Augmented Reality for AN Accessible Cultural Heritage. Digitization, 3d Printing and Sensors for AN Audio-Tactile Experience

    NASA Astrophysics Data System (ADS)

    D'Agnano, F.; Balletti, C.; Guerra, F.; Vernier, P.

    2015-02-01

    Tooteko is a smart ring that allows to navigate any 3D surface with your finger tips and get in return an audio content that is relevant in relation to the part of the surface you are touching in that moment. Tooteko can be applied to any tactile surface, object or sheet. However, in a more specific domain, it wants to make traditional art venues accessible to the blind, while providing support to the reading of the work for all through the recovery of the tactile dimension in order to facilitate the experience of contact with art that is not only "under glass." The system is made of three elements: a high-tech ring, a tactile surface tagged with NFC sensors, and an app for tablet or smartphone. The ring detects and reads the NFC tags and, thanks to the Tooteko app, communicates in wireless mode with the smart device. During the tactile navigation of the surface, when the finger reaches a hotspot, the ring identifies the NFC tag and activates, through the app, the audio track that is related to that specific hotspot. Thus a relevant audio content relates to each hotspot. The production process of the tactile surfaces involves scanning, digitization of data and 3D printing. The first experiment was modelled on the facade of the church of San Michele in Isola, made by Mauro Codussi in the late fifteenth century, and which marks the beginning of the Renaissance in Venice. Due to the absence of recent documentation on the church, the Correr Museum asked the Laboratorio di Fotogrammetria to provide it with the aim of setting up an exhibition about the order of the Camaldolesi, owners of the San Michele island and church. The Laboratorio has made the survey of the facade through laser scanning and UAV photogrammetry. The point clouds were the starting point for prototypation and 3D printing on different supports. The idea of the integration between a 3D printed tactile surface and sensors was born as a final thesis project at the Postgraduate Mastercourse in Digital

  16. Optimization of culture conditions for Gardnerella vaginalis biofilm formation.

    PubMed

    Machado, Daniela; Palmeira-de-Oliveira, Ana; Cerca, Nuno

    2015-11-01

    Bacterial vaginosis is the leading vaginal disorder in women in reproductive age. Although bacterial vaginosis is related with presence of a biofilm composed predominantly by Gardnerella vaginalis, there has not been a detailed information addressing the environmental conditions that influence the biofilm formation of this bacterial species. Here, we evaluated the influence of some common culture conditions on G. vaginalis biofilm formation, namely inoculum concentration, incubation period, feeding conditions and culture medium composition. Our results showed that culture conditions strongly influenced G. vaginalis biofilm formation and that biofilm formation was enhanced when starting the culture with a higher inoculum, using a fed-batch system and supplementing the growth medium with maltose. PMID:26381661

  17. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    PubMed Central

    Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

  18. Modeling and Accuracy Assessment for 3D-VIRTUAL Reconstruction in Cultural Heritage Using Low-Cost Photogrammetry: Surveying of the "santa MARÍA Azogue" Church's Front

    NASA Astrophysics Data System (ADS)

    Robleda Prieto, G.; Pérez Ramos, A.

    2015-02-01

    Sometimes it could be difficult to represent "on paper" an architectural idea, a solution, a detail or a newly created element, depending on the complexity what it want be conveyed through its graphical representation but it may be even harder to represent the existing reality. (a building, a detail,...), at least with an acceptable degree of definition and accuracy. As a solution to this hypothetical problem, this paper try to show a methodology to collect measure data by combining different methods or techniques, to obtain the characteristic geometry of architectonic elements, especially in those highly decorated and/or complex geometry, as well as to assess the accuracy of the results obtained, but in an accuracy level enough and not very expensive costs. In addition, we can obtain a 3D recovery model that allows us a strong support, beyond point clouds obtained through another more expensive methods as using laser scanner, to obtain orthoimages. This methodology was used in the study case of the 3D-virtual reconstruction of a main medieval church façade because of the geometrical complexity in many elements as the existing main doorway with archivolts and many details, as well as the rose window located above it so it's inaccessible due to the height.

  19. 3D microscope

    NASA Astrophysics Data System (ADS)

    Iizuka, Keigo

    2008-02-01

    In order to circumvent the fact that only one observer can view the image from a stereoscopic microscope, an attachment was devised for displaying the 3D microscopic image on a large LCD monitor for viewing by multiple observers in real time. The principle of operation, design, fabrication, and performance are presented, along with tolerance measurements relating to the properties of the cellophane half-wave plate used in the design.

  20. Biocompatible 3D Matrix with Antimicrobial Properties.

    PubMed

    Ion, Alberto; Andronescu, Ecaterina; Rădulescu, Dragoș; Rădulescu, Marius; Iordache, Florin; Vasile, Bogdan Ștefan; Surdu, Adrian Vasile; Albu, Madalina Georgiana; Maniu, Horia; Chifiriuc, Mariana Carmen; Grumezescu, Alexandru Mihai; Holban, Alina Maria

    2016-01-01

    The aim of this study was to develop, characterize and assess the biological activity of a new regenerative 3D matrix with antimicrobial properties, based on collagen (COLL), hydroxyapatite (HAp), β-cyclodextrin (β-CD) and usnic acid (UA). The prepared 3D matrix was characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Microscopy (FT-IRM), Transmission Electron Microscopy (TEM), and X-ray Diffraction (XRD). In vitro qualitative and quantitative analyses performed on cultured diploid cells demonstrated that the 3D matrix is biocompatible, allowing the normal development and growth of MG-63 osteoblast-like cells and exhibited an antimicrobial effect, especially on the Staphylococcus aureus strain, explained by the particular higher inhibitory activity of usnic acid (UA) against Gram positive bacterial strains. Our data strongly recommend the obtained 3D matrix to be used as a successful alternative for the fabrication of three dimensional (3D) anti-infective regeneration matrix for bone tissue engineering. PMID:26805790

  1. Microfibrillated cellulose sheets coating oxygen-permeable PDMS membranes induce rat hepatocytes 3D aggregation into stably-attached 3D hemispheroids.

    PubMed

    Evenou, Fanny; Couderc, Sandrine; Kim, Beomjoon; Fujii, Teruo; Sakai, Yasuyuki

    2011-01-01

    Here we report the use of natural, chemically-unmodified, microfibrillated cellulose (MFC) as a matrix for hepatocyte culture. We developed an original cell-culture design composed of a thin 3D-microstructured fibrous substrate consisting of a MFC sheet coating a highly O(2)-permeable polydimethylsiloxane (PDMS) membrane. The MFC-coated PDMS membranes were obtained according to a simple process where cellulose fibres were deposited from an aqueous suspension on the PDMS surfaces and the films were dried under mild conditions. To enable oxygen diffusion through the membranes, they were assembled on bottomless frames ('O(2)+' condition). Rat hepatocytes primary-cultured on such MFC-PDMS membranes quickly organized themselves into large hemispherical 3D aggregates which were tightly anchored to the MFC sheets. In contrast, hepatocytes cultured on smooth PDMS membranes in the O(2)+ system (O(2)+, PDMS) organized into unstable 2D monolayers which easily detached from the surfaces. Hepatocyte 3D cultures obtained on MFC-PDMS membranes exhibited higher liver-specific functions over a 2-week culture period, as assessed by both the higher albumin secretion and urea synthesis rate. The MFC-PDMS membranes appear suitable for obtaining stably-attached and functional hepatocyte 3D cultures and appear interesting for drug/chemical screenings in a microplate format, but also for microfluidic applications. PMID:20626957

  2. Defining cell culture conditions to improve human norovirus infectivity assays.

    PubMed

    Straub, T M; Hutchison, J R; Bartholomew, R A; Valdez, C O; Valentine, N B; Dohnalkova, A; Ozanich, R M; Bruckner-Lea, C J

    2013-01-01

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus. PMID:23306266

  3. Deducing the subsurface geological conditions and structural framework of the NE Gulf of Suez area, using 2-D and 3-D seismic data

    NASA Astrophysics Data System (ADS)

    Zahra, Hesham Shaker; Nakhla, Adel Mokhles

    2015-06-01

    An interpretation of the seismic data of Ras Budran and Abu Zenima oil fields, northern central Gulf of Suez, is carried out to evaluate its subsurface tectonic setting. The structural configuration, as well as the tectonic features of the concerned area is criticized through the study of 2D and 3D seismic data interpretation with the available geological data, in which the geo-seismic depth maps for the main interesting levels (Kareem, Nukhul, Matulla, Raha and Nubia Formations) are depicted. Such maps reflect that, the Miocene structure of Ras Budran area is a nearly NE-SW trending anticlinal feature, which broken into several panels by a set of NWSE and NE-SW trending faults. The Pre-Miocene structure of the studied area is very complex, where Ras Budran area consists of step faults down stepping to the south and southwest, which have been subjected to cross faults of NE-SW trend with lateral and vertical displacements.

  4. Microscale screening systems for 3D cellular microenvironments: platforms, advances, and challenges

    PubMed Central

    Montanez-Sauri, Sara I.; Beebe, David J.; Sung, Kyung Eun

    2015-01-01

    The increasing interest in studying cells using more in vivo-like three-dimensional (3D) microenvironments has created a need for advanced 3D screening platforms with enhanced functionalities and increased throughput. 3D screening platforms that better mimic in vivo microenvironments with enhanced throughput would provide more in-depth understanding of the complexity and heterogeneity of microenvironments. The platforms would also better predict the toxicity and efficacy of potential drugs in physiologically relevant conditions. Traditional 3D culture models (e.g. spinner flasks, gyratory rotation devices, non-adhesive surfaces, polymers) were developed to create 3D multicellular structures. However, these traditional systems require large volumes of reagents and cells, and are not compatible with high throughput screening (HTS) systems. Microscale technology offers the miniaturization of 3D cultures and allows efficient screening of various conditions. This review will discuss the development, most influential works, and current advantages and challenges of microscale culture systems for screening cells in 3D microenvironments. PMID:25274061

  5. Alternation of adriamycin penetration kinetics in MCF-7 cells from 2D to 3D culture based on P-gp expression through the Chk2/p53/NF-κB pathway.

    PubMed

    Lu, Meng; Zhou, Fang; Hao, Kun; Liu, Jiali; Chen, Qianying; Ni, Ping; Zhou, Honghao; Wang, Guangji; Zhang, Jingwei

    2015-01-15

    Monolayer cells are largely different from tumor masses, and might misguide drug screenings. 3D in vitro cell culture models simulate the characteristics of tumor masses in vivo and have recently been used in many studies of anti-cancer drugs. Among various 3D cell culture models, multi-cellular layer (MCL) models allow for the direct quantitative assessment of the penetration of chemotherapeutic agents through solid tissue environments without requiring the use of fluorescently labeled drugs or imaging molecules. Therefore, in our present study, a 3D-no base and embedded MCF-7 MCL model was successfully developed for a 14-day culture. Over time, its thickness and cell layers increased and exhibited highly proliferative properties and drug resistance to adriamycin (ADR) with markedly elevated IC50 values. Meanwhile, G2/M stage cycle arrest was also observed, which likely up-regulated P-gp expression through the Chk2/p53/NF-κB pathway. The elevated P-gp expression altered the ADR penetration kinetics in MCF-7 MCLs in vitro by accelerating the apparent penetration of ADR through the intercellular spaces of the MCLs. Additionally, a decreased ADR retention within tumor cells was observed, but could be significantly reversed by a P-gp inhibitor. The attenuated ADR retention in the deeper cells of tumor masses was confirmed in xenografted mice in vivo. This phenomenon could be elucidated by the mathematical modeling of penetration kinetics parameters. Our study provided a new model that evaluated and improved the quantification of the drug penetration kinetics, revealed the relationship between P-gp and drug penetration through tumor masses, and suggested the potential molecular mechanisms. PMID:25478729

  6. Multiviewer 3D monitor

    NASA Astrophysics Data System (ADS)

    Kostrzewski, Andrew A.; Aye, Tin M.; Kim, Dai Hyun; Esterkin, Vladimir; Savant, Gajendra D.

    1998-09-01

    Physical Optics Corporation has developed an advanced 3-D virtual reality system for use with simulation tools for training technical and military personnel. This system avoids such drawbacks of other virtual reality (VR) systems as eye fatigue, headaches, and alignment for each viewer, all of which are due to the need to wear special VR goggles. The new system is based on direct viewing of an interactive environment. This innovative holographic multiplexed screen technology makes it unnecessary for the viewer to wear special goggles.

  7. 3D Audio System

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Ames Research Center research into virtual reality led to the development of the Convolvotron, a high speed digital audio processing system that delivers three-dimensional sound over headphones. It consists of a two-card set designed for use with a personal computer. The Convolvotron's primary application is presentation of 3D audio signals over headphones. Four independent sound sources are filtered with large time-varying filters that compensate for motion. The perceived location of the sound remains constant. Possible applications are in air traffic control towers or airplane cockpits, hearing and perception research and virtual reality development.

  8. Optimal Culture Conditions for Mycelial Growth of Lignosus rhinocerus

    PubMed Central

    Siti Murni, M.J.; Fauzi, D.; Abas Mazni, O.; Saleh, N.M.

    2011-01-01

    Lignosus rhinocerus is a macrofungus that belongs to Polyporaceae and is native to tropical regions. This highly priced mushroom has been used as folk medicine to treat diseases by indigenous people. As a preliminary study to develop a culture method for edible mushrooms, the cultural characteristics of L. rhinocerus were investigated in a range of culture media under different environmental conditions. Mycelial growth of this mushroom was compared on culture media composed of various carbon and nitrogen sources in addition to C/N ratios. The optimal conditions for mycelial growth were 30℃ at pH 6 and 7. Rapid mycelial growth of L. rhinocerus was observed on glucose-peptone and yeast extract peptone dextrose media. Carbon and nitrogen sources promoting mycelial growth of L. rhinocerus were glucose and potassium nitrate, respectively. The optimum C/N ratio was approximately 10 : 1 using 2% glucose supplemented as a carbon source in the basal media. PMID:22783083

  9. A 3D-Printed Oxygen Control Insert for a 24-Well Plate

    PubMed Central

    Brennan, Martin D.; Rexius-Hall, Megan L.; Eddington, David T.

    2015-01-01

    3D printing has emerged as a method for directly printing complete microfluidic devices, although printing materials have been limited to oxygen-impermeable materials. We demonstrate the addition of gas permeable PDMS (Polydimethylsiloxane) membranes to 3D-printed microfluidic devices as a means to enable oxygen control cell culture studies. The incorporation of a 3D-printed device and gas-permeable membranes was demonstrated on a 24-well oxygen control device for standard multiwell plates. The direct printing allows integrated distribution channels and device geometries not possible with traditional planar lithography. With this device, four different oxygen conditions were able to be controlled, and six wells were maintained under each oxygen condition. We demonstrate enhanced transcription of the gene VEGFA (vascular endothelial growth factor A) with decreasing oxygen levels in human lung adenocarcinoma cells. This is the first 3D-printed device incorporating gas permeable membranes to facilitate oxygen control in cell culture. PMID:26360882

  10. Influence of the laser assisted fabricated 3D porous scaffolds from bioceramoplasts of micron and nano sizes on culture of MMSC

    NASA Astrophysics Data System (ADS)

    Shishkovsky, I.; Volchkov, S.

    2013-11-01

    The objective of the investigation was to test the biocompatibility of 3D porous biopolymer matrices (tissue-cellular scaffolds), made of biocompatible and bioresorbable polymers (polycarbonate, polyetheretherketone /PEEK/, polycaprolactone), including the materials with biocompatible oxide ceramics additive (TiO2, Al2O3, ZrO2 and hydroxyapatite) of micron and nano sizes, for tissue-engineering purposes. The porous samples were prepared via a layer-by-layer SLS method. The surface microstructures and their roughness were analyzed by the optical microscopy equipped with the cell analysis software. The cellular morphology, proliferative activity and adhesion of the polymeric and ceramopolymeric matrices were the subjects for comparison. The study showed that all the tested materials posessed biocompatible properties. The experimentally estimated cell duplication speed per day turned out to be maximal for polycarbonate (0.279 duplications per day) and for PEEK + Al2O3 = 3:1 group (0.30 dupl/day) against 0.387 dupl/day for the reference sample and 0.270 dupl/day for the group of cells placed close to the pure titanium samples.

  11. 3D Surgical Simulation

    PubMed Central

    Cevidanes, Lucia; Tucker, Scott; Styner, Martin; Kim, Hyungmin; Chapuis, Jonas; Reyes, Mauricio; Proffit, William; Turvey, Timothy; Jaskolka, Michael

    2009-01-01

    This paper discusses the development of methods for computer-aided jaw surgery. Computer-aided jaw surgery allows us to incorporate the high level of precision necessary for transferring virtual plans into the operating room. We also present a complete computer-aided surgery (CAS) system developed in close collaboration with surgeons. Surgery planning and simulation include construction of 3D surface models from Cone-beam CT (CBCT), dynamic cephalometry, semi-automatic mirroring, interactive cutting of bone and bony segment repositioning. A virtual setup can be used to manufacture positioning splints for intra-operative guidance. The system provides further intra-operative assistance with the help of a computer display showing jaw positions and 3D positioning guides updated in real-time during the surgical procedure. The CAS system aids in dealing with complex cases with benefits for the patient, with surgical practice, and for orthodontic finishing. Advanced software tools for diagnosis and treatment planning allow preparation of detailed operative plans, osteotomy repositioning, bone reconstructions, surgical resident training and assessing the difficulties of the surgical procedures prior to the surgery. CAS has the potential to make the elaboration of the surgical plan a more flexible process, increase the level of detail and accuracy of the plan, yield higher operative precision and control, and enhance documentation of cases. Supported by NIDCR DE017727, and DE018962 PMID:20816308

  12. Arginine–glycine–aspartic acid–polyethylene glycol–polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture

    PubMed Central

    Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

    2016-01-01

    The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine–glycine–aspartic acid (RGD) peptide. Our studies demonstrate that RGD–polyethylene glycol (PEG)–PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT–MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system. PMID:27621619

  13. Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

    PubMed

    Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

    2016-01-01

    The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system. PMID:27621619

  14. Modular, pumpless body-on-a-chip platform for the co-culture of GI tract epithelium and 3D primary liver tissue.

    PubMed

    Esch, Mandy B; Ueno, Hidetaka; Applegate, Dawn R; Shuler, Michael L

    2016-07-01

    We have developed an expandable modular body-on-a-chip system that allows for a plug-and-play approach with several in vitro tissues. The design consists of single-organ chips that are combined with each other to yield a multi-organ body-on-a-chip system. Fluidic flow through the organ chips is driven via gravity and controlled passively via hydraulic resistances of the microfluidic channel network. Such pumpless body-on-a-chip devices are inexpensive and easy to use. We tested the device by culturing GI tract tissue and liver tissue within the device. Integrated Ag/AgCl electrodes were used to measure the resistance across the GI tract cell layer. The transepithelial resistance (TEER) reached values between 250 to 650 Ω cm(2) throughout the 14 day co-culture period. These data indicate that the GI tract cells retained their viability and the GI tract layer as a whole retained its barrier function. Throughout the 14 day co-culture period we measured low amounts of aspartate aminotransferase (AST, ∼10-17.5 U L(-1)), indicating low rates of liver cell death. Metabolic rates of hepatocytes were comparable to those of hepatocytes in single-organ fluidic cell culture systems (albumin production ranged between 3-6 μg per day per million hepatocytes and urea production ranged between 150-200 μg per day per million hepatocytes). Induced CYP activities were higher than previously measured with microfluidic liver only systems. PMID:27332143

  15. From "sapienza" to "sapienza, State Archives in Rome". a Looping Effect Bringing Back to the Original Source Comunication and Culture by Innovative and Low Cost 3d Surveying, Imaging Systems and GIS Applications

    NASA Astrophysics Data System (ADS)

    Paolini, P.; Forti, G.; Catalani, G.; Lucchetti, S.; Menghini, A.; Mirandola, A.; Pistacchio, S.; Porzia, U.; Roberti, M.

    2016-04-01

    High Quality survey models, realized by multiple Low Cost methods and technologies, as a container to sharing Cultural and Archival Heritage, this is the aim guiding our research, here described in its primary applications. The SAPIENZA building, a XVI century masterpiece that represented the first unified headquarters of University in Rome, plays since year 1936, when the University moved to its newly edified campus, the role of the main venue for the State Archives. By the collaboration of a group of students of the Architecture Faculty, some integrated survey methods were applied on the monument with success. The beginning was the topographic survey, creating a reference on ground and along the monument for the upcoming applications, a GNNS RTK survey followed georeferencing points on the internal courtyard. Dense stereo matching photogrammetry is nowadays an accepted method for generating 3D survey models, accurate and scalable; it often substitutes 3D laser scanning for its low cost, so that it became our choice. Some 360° shots were planned for creating panoramic views of the double portico from the courtyard, plus additional single shots of some lateral spans and of pillars facing the court, as a single operation with a double finality: to create linked panotours with hotspots to web-linked databases, and 3D textured and georeferenced surface models, allowing to study the harmonic proportions of the classical architectural order. The use of free web Gis platforms, to load the work in Google Earth and the realization of low cost 3D prototypes of some representative parts, has been even performed.

  16. Primary cancer cell culture: mammary-optimized vs conditional reprogramming.

    PubMed

    Alamri, Ahmad M; Kang, Keunsoo; Groeneveld, Svenja; Wang, Weisheng; Zhong, Xiaogang; Kallakury, Bhaskar; Hennighausen, Lothar; Liu, Xuefeng; Furth, Priscilla A

    2016-07-01

    The impact of different culture conditions on biology of primary cancer cells is not always addressed. Here, conditional reprogramming (CRC) was compared with mammary-optimized EpiCult-B (EpiC) for primary mammary epithelial cell isolation and propagation, allograft generation, and genome-wide transcriptional consequences using cancer and non-cancer mammary tissue from mice with different dosages of Brca1 and p53 Selective comparison to DMEM was included. Primary cultures were established with all three media, but CRC was most efficient for initial isolation (P<0.05). Allograft development was faster using cells grown in EpiC compared with CRC (P<0.05). Transcriptome comparison of paired CRC and EpiC cultures revealed 1700 differentially expressed genes by passage 20. CRC promoted Trp53 gene family upregulation and increased expression of epithelial differentiation genes, whereas EpiC elevated expression of epithelial-mesenchymal transition genes. Differences did not persist in allografts where both methods yielded allografts with relatively similar transcriptomes. Restricting passage (<7) reduced numbers of differentially expressed genes below 50. In conclusion, CRC was most efficient for initial cell isolation but EpiC was quicker for allograft generation. The extensive culture-specific gene expression patterns that emerged with longer passage could be limited by reducing passage number when both culture transcriptomes were equally similar to that of the primary tissue. Defining impact of culture condition and passage on the transcriptome of primary cells could assist experimental design and interpretation. For example, differences that appear with passage and culture condition are potentially exploitable for comparative studies targeting specific biological networks in different transcriptional environments. PMID:27267121

  17. 3D Printed Bionic Ears

    PubMed Central

    Mannoor, Manu S.; Jiang, Ziwen; James, Teena; Kong, Yong Lin; Malatesta, Karen A.; Soboyejo, Winston O.; Verma, Naveen; Gracias, David H.; McAlpine, Michael C.

    2013-01-01

    The ability to three-dimensionally interweave biological tissue with functional electronics could enable the creation of bionic organs possessing enhanced functionalities over their human counterparts. Conventional electronic devices are inherently two-dimensional, preventing seamless multidimensional integration with synthetic biology, as the processes and materials are very different. Here, we present a novel strategy for overcoming these difficulties via additive manufacturing of biological cells with structural and nanoparticle derived electronic elements. As a proof of concept, we generated a bionic ear via 3D printing of a cell-seeded hydrogel matrix in the precise anatomic geometry of a human ear, along with an intertwined conducting polymer consisting of infused silver nanoparticles. This allowed for in vitro culturing of cartilage tissue around an inductive coil antenna in the ear, which subsequently enables readout of inductively-coupled signals from cochlea-shaped electrodes. The printed ear exhibits enhanced auditory sensing for radio frequency reception, and complementary left and right ears can listen to stereo audio music. Overall, our approach suggests a means to intricately merge biologic and nanoelectronic functionalities via 3D printing. PMID:23635097

  18. 3D printed bionic ears.

    PubMed

    Mannoor, Manu S; Jiang, Ziwen; James, Teena; Kong, Yong Lin; Malatesta, Karen A; Soboyejo, Winston O; Verma, Naveen; Gracias, David H; McAlpine, Michael C

    2013-06-12

    The ability to three-dimensionally interweave biological tissue with functional electronics could enable the creation of bionic organs possessing enhanced functionalities over their human counterparts. Conventional electronic devices are inherently two-dimensional, preventing seamless multidimensional integration with synthetic biology, as the processes and materials are very different. Here, we present a novel strategy for overcoming these difficulties via additive manufacturing of biological cells with structural and nanoparticle derived electronic elements. As a proof of concept, we generated a bionic ear via 3D printing of a cell-seeded hydrogel matrix in the anatomic geometry of a human ear, along with an intertwined conducting polymer consisting of infused silver nanoparticles. This allowed for in vitro culturing of cartilage tissue around an inductive coil antenna in the ear, which subsequently enables readout of inductively-coupled signals from cochlea-shaped electrodes. The printed ear exhibits enhanced auditory sensing for radio frequency reception, and complementary left and right ears can listen to stereo audio music. Overall, our approach suggests a means to intricately merge biologic and nanoelectronic functionalities via 3D printing. PMID:23635097

  19. 3D polarimetric purity

    NASA Astrophysics Data System (ADS)

    Gil, José J.; San José, Ignacio

    2010-11-01

    From our previous definition of the indices of polarimetric purity for 3D light beams [J.J. Gil, J.M. Correas, P.A. Melero and C. Ferreira, Monogr. Semin. Mat. G. de Galdeano 31, 161 (2004)], an analysis of their geometric and physical interpretation is presented. It is found that, in agreement with previous results, the first parameter is a measure of the degree of polarization, whereas the second parameter (called the degree of directionality) is a measure of the mean angular aperture of the direction of propagation of the corresponding light beam. This pair of invariant, non-dimensional, indices of polarimetric purity contains complete information about the polarimetric purity of a light beam. The overall degree of polarimetric purity is obtained as a weighted quadratic average of the degree of polarization and the degree of directionality.

  20. 3D field harmonics

    SciTech Connect

    Caspi, S.; Helm, M.; Laslett, L.J.

    1991-03-30

    We have developed an harmonic representation for the three dimensional field components within the windings of accelerator magnets. The form by which the field is presented is suitable for interfacing with other codes that make use of the 3D field components (particle tracking and stability). The field components can be calculated with high precision and reduced cup time at any location (r,{theta},z) inside the magnet bore. The same conductor geometry which is used to simulate line currents is also used in CAD with modifications more readily available. It is our hope that the format used here for magnetic fields can be used not only as a means of delivering fields but also as a way by which beam dynamics can suggest correction to the conductor geometry. 5 refs., 70 figs.

  1. 'Bonneville' in 3-D!

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The Mars Exploration Rover Spirit took this 3-D navigation camera mosaic of the crater called 'Bonneville' after driving approximately 13 meters (42.7 feet) to get a better vantage point. Spirit's current position is close enough to the edge to see the interior of the crater, but high enough and far enough back to get a view of all of the walls. Because scientists and rover controllers are so pleased with this location, they will stay here for at least two more martian days, or sols, to take high resolution panoramic camera images of 'Bonneville' in its entirety. Just above the far crater rim, on the left side, is the rover's heatshield, which is visible as a tiny reflective speck.

  2. Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D) human cell culture model of oral mucositis

    PubMed Central

    Lambros, Maria P.; DeSalvo, Michael K.; Moreno, Jonathan; Mulamalla, Hari Chandana; Kondapalli, Lavanya

    2015-01-01

    Cancer patients who receive radiation are often afflicted by oral mucositis, a debilitating disease, characterized by mouth sores and difficulty in swallowing. Oftentimes, cancer patients afflicted with mucositis must stop life-saving therapies. Thus it is very important to prevent mucositis before it develops. Using a validated organotypic model of human oral mucosa, a 3-dimensional cell culture model of human oral keratinocytes, it has been shown that a mixture (NAC–QYD) of N-acetyl cysteine (NAC) and a traditional Chinese medicine, Qingre Liyan decoction (QYD), prevented radiation damage (Lambros et al., 2014). Here we provide detailed methods and analysis of microarray data for non-irradiated and irradiated human oral mucosal tissue with and without pretreatment with NAC, QYD and NAC-QYD. The microarray data been deposited in Gene Expression Omnibus (GEO): GSE62397. These data can be used to further elucidate the mechanisms of irradiation damage in oral mucosa and its prevention. PMID:26697327

  3. Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D) human cell culture model of oral mucositis.

    PubMed

    Lambros, Maria P; DeSalvo, Michael K; Moreno, Jonathan; Mulamalla, Hari Chandana; Kondapalli, Lavanya

    2015-12-01

    Cancer patients who receive radiation are often afflicted by oral mucositis, a debilitating disease, characterized by mouth sores and difficulty in swallowing. Oftentimes, cancer patients afflicted with mucositis must stop life-saving therapies. Thus it is very important to prevent mucositis before it develops. Using a validated organotypic model of human oral mucosa, a 3-dimensional cell culture model of human oral keratinocytes, it has been shown that a mixture (NAC-QYD) of N-acetyl cysteine (NAC) and a traditional Chinese medicine, Qingre Liyan decoction (QYD), prevented radiation damage (Lambros et al., 2014). Here we provide detailed methods and analysis of microarray data for non-irradiated and irradiated human oral mucosal tissue with and without pretreatment with NAC, QYD and NAC-QYD. The microarray data been deposited in Gene Expression Omnibus (GEO): GSE62397. These data can be used to further elucidate the mechanisms of irradiation damage in oral mucosa and its prevention. PMID:26697327

  4. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    NASA Astrophysics Data System (ADS)

    Shamloo, Amir; Amirifar, Leyla

    2016-01-01

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies.

  5. Biomek Cell Workstation: A Flexible System for Automated 3D Cell Cultivation.

    PubMed

    Lehmann, R; Gallert, C; Roddelkopf, T; Junginger, S; Thurow, K

    2016-08-01

    The shift from 2D cultures to 3D cultures enables improvement in cell culture research due to better mimicking of in vivo cell behavior and environmental conditions. Different cell lines and applications require altered 3D constructs. The automation of the manufacturing and screening processes can advance the charge stability, quality, repeatability, and precision. In this study we integrated the automated production of three 3D cell constructs (alginate beads, spheroid cultures, pellet cultures) using the Biomek Cell Workstation and compared them with the traditional manual methods and their consequent bioscreening processes (proliferation, toxicity; days 14 and 35) using a high-throughput screening system. Moreover, the possible influence of antibiotics (penicillin/streptomycin) on the production and screening processes was investigated. The cytotoxicity of automatically produced 3D cell cultures (with and without antibiotics) was mainly decreased. The proliferation showed mainly similar or increased results for the automatically produced 3D constructs. We concluded that the traditional manual methods can be replaced by the automated processes. Furthermore, the formation, cultivation, and screenings can be performed without antibiotics to prevent possible effects. PMID:26203054

  6. Prominent rocks - 3D

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Many prominent rocks near the Sagan Memorial Station are featured in this image, taken in stereo by the Imager for Mars Pathfinder (IMP) on Sol 3. 3D glasses are necessary to identify surface detail. Wedge is at lower left; Shark, Half-Dome, and Pumpkin are at center. Flat Top, about four inches high, is at lower right. The horizon in the distance is one to two kilometers away.

    Mars Pathfinder is the second in NASA's Discovery program of low-cost spacecraft with highly focused science goals. The Jet Propulsion Laboratory, Pasadena, CA, developed and manages the Mars Pathfinder mission for NASA's Office of Space Science, Washington, D.C. JPL is an operating division of the California Institute of Technology (Caltech). The Imager for Mars Pathfinder (IMP) was developed by the University of Arizona Lunar and Planetary Laboratory under contract to JPL. Peter Smith is the Principal Investigator.

    Click below to see the left and right views individually. [figure removed for brevity, see original site] Left [figure removed for brevity, see original site] Right

  7. 'Diamond' in 3-D

    NASA Technical Reports Server (NTRS)

    2004-01-01

    This 3-D, microscopic imager mosaic of a target area on a rock called 'Diamond Jenness' was taken after NASA's Mars Exploration Rover Opportunity ground into the surface with its rock abrasion tool for a second time.

    Opportunity has bored nearly a dozen holes into the inner walls of 'Endurance Crater.' On sols 177 and 178 (July 23 and July 24, 2004), the rover worked double-duty on Diamond Jenness. Surface debris and the bumpy shape of the rock resulted in a shallow and irregular hole, only about 2 millimeters (0.08 inch) deep. The final depth was not enough to remove all the bumps and leave a neat hole with a smooth floor. This extremely shallow depression was then examined by the rover's alpha particle X-ray spectrometer.

    On Sol 178, Opportunity's 'robotic rodent' dined on Diamond Jenness once again, grinding almost an additional 5 millimeters (about 0.2 inch). The rover then applied its Moessbauer spectrometer to the deepened hole. This double dose of Diamond Jenness enabled the science team to examine the rock at varying layers. Results from those grindings are currently being analyzed.

    The image mosaic is about 6 centimeters (2.4 inches) across.

  8. A 3D Toolbox to Enhance Physiological Relevance of Human Tissue Models.

    PubMed

    Picollet-D'hahan, Nathalie; Dolega, Monika E; Liguori, Lavinia; Marquette, Christophe; Le Gac, Séverine; Gidrol, Xavier; Martin, Donald K

    2016-09-01

    We discuss the current challenges and future prospects of flow-based organoid models and 3D self-assembling scaffolds. The existing paradigm of 3D culture suffers from a lack of control over organoid size and shape; can be an obstacle for cell harvesting and extended cellular and molecular analysis; and does not provide access to the function of exocrine glands. Moreover, existing organ-on-chip models are mostly composed of 2D extracellular matrix (ECM)-coated elastomeric membranes that do not mimic real organ architectures. A new comprehensive 3D toolbox for cell biology has emerged to address some of these issues. Advances in microfabrication and cell-culturing approaches enable the engineering of sophisticated models that mimic organ 3D architectures and physiological conditions, while supporting flow-based drug screening and secretomics-based diagnosis. PMID:27497676

  9. The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes - A genome-wide study in UVB irradiated organotypic 3D cultures.

    PubMed

    Rauhala, Leena; Hämäläinen, Lasse; Dunlop, Thomas W; Pehkonen, Petri; Bart, Geneviève; Kokkonen, Maarit; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-12-25

    The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation. PMID:26391144

  10. Combining depth analysis with surface morphology analysis to analyse the prehistoric painted pottery from Majiayao Culture by confocal 3D-XRF

    NASA Astrophysics Data System (ADS)

    Yi, Longtao; Liu, Zhiguo; Wang, Kai; Lin, Xue; Chen, Man; Peng, Shiqi; Yang, Kui; Wang, Jinbang

    2016-04-01

    The Majiayao Culture (3300 BC-2900 BC) formed one of the three painted pottery centres of the Yellow River basin, China, in prehistoric times. Painted pottery from this period is famous for its exquisite workmanship and meticulous painting. Studying the layer structure and element distribution of the paint on the pottery is conducive to investigating its workmanship, which is important for archaeological research. However, the most common analysis methods are destructive. To investigate the layers of paint on the pottery nondestructively, a confocal three-dimensional micro-X-ray fluorescence set-up combined with two individual polycapillary lenses has been used to analyse two painted pottery fragments. Nondestructive elemental depth analyses and surface topographic analysis were performed. The elemental depth profiles of Mn, Fe and Ca obtained from these measurements were consistent with those obtained using an optical microscope. The depth profiles show that there are layer structures in two samples. The images show that the distribution of Ca is approximately homogeneous in both painted and unpainted regions. In contrast, Mn appeared only in the painted regions. Meanwhile, the distributions of Fe in the painted and unpainted regions were not the same. The surface topographic shows that the pigment of dark-brown region was coated above the brown region. These conclusions allowed the painting process to be inferred.

  11. A novel injectable thermoresponsive and cytocompatible gel of poly(N-isopropylacrylamide) with layered double hydroxides facilitates siRNA delivery into chondrocytes in 3D culture.

    PubMed

    Yang, Hsiao-yin; van Ee, Renz J; Timmer, Klaas; Craenmehr, Eric G M; Huang, Julie H; Öner, F Cumhur; Dhert, Wouter J A; Kragten, Angela H M; Willems, Nicole; Grinwis, Guy C M; Tryfonidou, Marianna A; Papen-Botterhuis, Nicole E; Creemers, Laura B

    2015-09-01

    Hybrid hydrogels composed of poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) are presented in this study as novel injectable and thermoresponsive materials for siRNA delivery, which could specifically target several negative regulators of tissue homeostasis in cartilaginous tissues. Effectiveness of siRNA transfection using pNIPAAM formulated with either MgAl-LDH or MgFe-LDH platelets was investigated using osteoarthritic chondrocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing. No significant adverse effects of pNIPAAM/LDH hydrogels on cell viability were noticed. Cellular uptake of fluorescently labeled siRNA was greatly enhanced (>75%) in pNIPAAM/LDH hydrogel constructs compared to alginate, hyaluronan and fibrin gels, and was absent in pNIPAAM hydrogel without LDH platelets. When using siRNA against GAPDH, 82-98% reduction of gene expression was found in both types of pNIPAAM/LDH hydrogel constructs after 6 days of culturing. In the pNIPAAM/MgAl-LDH hybrid hydrogel, 80-95% of GAPDH enzyme activity was reduced in parallel with gene. Our findings show that the combination of a cytocompatible hydrogel and therapeutic RNA oligonucleotides is feasible. Thus it might hold promise in treating degeneration of cartilaginous tissues by providing supporting scaffolds for cells and interference with locally produced degenerative factors. PMID:26022968

  12. Incompressible limit of all-time solutions to 3-D full Navier-Stokes equations for perfect gas with well-prepared initial condition

    NASA Astrophysics Data System (ADS)

    Ren, Dandan; Ou, Yaobin

    2016-08-01

    In this paper, we prove the incompressible limit of all-time strong solutions to the three-dimensional full compressible Navier-Stokes equations. Here the velocity field and temperature satisfy the Dirichlet boundary condition and convective boundary condition, respectively. The uniform estimates in both the Mach number {ɛin(0,overline{ɛ}]} and time {tin[0,∞)} are established by deriving a differential inequality with decay property, where {overline{ɛ} in(0,1]} is a constant. Based on these uniform estimates, the global solution of full compressible Navier-Stokes equations with "well-prepared" initial conditions converges to the one of isentropic incompressible Navier-Stokes equations as the Mach number goes to zero.

  13. The Educational Value of Visual Cues and 3D-Representational Format in a Computer Animation under Restricted and Realistic Conditions

    ERIC Educational Resources Information Center

    Huk, Thomas; Steinke, Mattias; Floto, Christian

    2010-01-01

    Within the framework of cognitive learning theories, instructional design manipulations have primarily been investigated under tightly controlled laboratory conditions. We carried out two experiments, where the first experiment was conducted in a restricted system-paced setting and is therefore in line with the majority of empirical studies in the…

  14. Preferential upward flow in soils: A 3D comparison of modeled and ERT-derived data from a salt tracer experiment under evaporation conditions

    NASA Astrophysics Data System (ADS)

    Bechtold, Michel; Vanderborght, Jan; Herbst, Michael; Weihermueller, Lutz; Kasteel, Roy; Ippisch, Olaf; Guenther, Thomas; Vereecken, Harry

    2010-05-01

    Upward water flow induced by evaporation or groundwater level rise can cause soil salinization and transport of contaminants to the soil surface. A limitation for the prediction of upward transport using numerical models might be an incomplete process description of this transport within the models, especially under consideration of heterogeneous structures. In contrast to infiltration conditions, few experimental datasets of transport under upward flow conditions that can be used to test existing models exist. Therefore, we studied upward transport at the pedon-scale in a laboratory soil with a defined heterogeneity and controlled upper and lower boundary conditions. A second aim was the assessment of the potential of Electrical Resistivity Tomography (ERT) to image and characterize upward transport and to use these temporal and spatial highly resolved experimental data to validate current model approaches. Using stochastic simulation, we designed a laboratory soil composed of three materials, which represent a correlated indicator field with horizontal and vertical heterogeneity. A salt tracer experiment was performed over 40 days with steady-state upward flow. Constant evaporation conditions were established using an air-conditioning chamber. A constant water level with the tracer solution was imposed at the lower boundary. ERT results showed solute mass flowing upwards along a few preferential pathways and accumulating heterogeneously at the soil surface. Three-dimensional numerical simulations based on Richards' and the convection-dispersion equation satisfactorily described solute transport in the lower part of the soil, whereas closer to the surface larger discrepancies occurred. On the experimental side, uncertainties in the petrophysical relationship and spatial smoothing inherent to the applied Occam-type smoothness constrained geophysical inversion contributed to observed deviations between ERT and model results. Comparing measured with modeled (using

  15. Task reports on developing techniques for scattering by 3D composite structures and to generate new solutions in diffraction theory using higher order boundary conditions

    NASA Technical Reports Server (NTRS)

    Volakis, John L.

    1990-01-01

    There are two tasks described in this report. First, an extension of a two dimensional formulation is presented for a three dimensional body of revolution. With the introduction of a Fourier expansion of the vector electric and magnetic fields, a coupled two dimensional system is generated and solved via the finite element method. An exact boundary condition is employed to terminate the mesh and the fast fourier transformation is used to evaluate the boundary integrals for low O(n) memory demand when an iterative solution algorithm is used. Second, the diffraction by a material discontinuity in a thick dielectric/ferrite layer is considered by modeling the layer as a distributed current sheet obeying generalized sheet transition conditions (GSTC's).

  16. Task reports on developing techniques for scattering by 3D composite structures and to generate new solutions in diffraction theory using higher order boundary conditions

    NASA Technical Reports Server (NTRS)

    Volakis, John L.

    1991-01-01

    There are two tasks described in this report. First, an extension of a two dimensional formulation is presented for a three dimensional body of revolution. A Fourier series expansion of the vector electric and magnetic fields is employed to reduce the dimensionality of the system, and an exact boundary condition is employed to terminate the mesh. The mesh termination boundary is chosen such that it leads to convolutional boundary operators for low O(n) memory demand. Second, rigorous uniform geometrical theory of diffraction (UTD) diffraction coefficients are presented for a coated convex cylinder simulated with generalized impedance boundary conditions. Ray solutions are obtained which remain valid in the transition region and reduce uniformly those in the deep lit and shadow regions. A uniform asymptotic solution is also presented for observations in the close vicinity of the cylinder.

  17. The psychology of the 3D experience

    NASA Astrophysics Data System (ADS)

    Janicke, Sophie H.; Ellis, Andrew

    2013-03-01

    With 3D televisions expected to reach 50% home saturation as early as 2016, understanding the psychological mechanisms underlying the user response to 3D technology is critical for content providers, educators and academics. Unfortunately, research examining the effects of 3D technology has not kept pace with the technology's rapid adoption, resulting in large-scale use of a technology about which very little is actually known. Recognizing this need for new research, we conducted a series of studies measuring and comparing many of the variables and processes underlying both 2D and 3D media experiences. In our first study, we found narratives within primetime dramas had the power to shift viewer attitudes in both 2D and 3D settings. However, we found no difference in persuasive power between 2D and 3D content. We contend this lack of effect was the result of poor conversion quality and the unique demands of 3D production. In our second study, we found 3D technology significantly increased enjoyment when viewing sports content, yet offered no added enjoyment when viewing a movie trailer. The enhanced enjoyment of the sports content was shown to be the result of heightened emotional arousal and attention in the 3D condition. We believe the lack of effect found for the movie trailer may be genre-related. In our final study, we found 3D technology significantly enhanced enjoyment of two video games from different genres. The added enjoyment was found to be the result of an increased sense of presence.

  18. In vivo ectopic bone formation by devitalized mineralized stem cell carriers produced under mineralizing culture condition.

    PubMed

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P; Schrooten, Jan

    2014-12-01

    Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography

  19. In Vivo Ectopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition

    PubMed Central

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P.

    2014-01-01

    Abstract Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43−) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano

  20. Defining cell culture conditions to improve human norovirus infectivity assays

    SciTech Connect

    Straub, Tim M.; Hutchison, Janine R.; Bartholomew, Rachel A.; Valdez, Catherine O.; Valentine, Nancy B.; Dohnalkova, Alice; Ozanich, Richard M.; Bruckner-Lea, Cindy J.

    2013-01-10

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

  1. 3-D Relativistic MHD Simulations

    NASA Astrophysics Data System (ADS)

    Nishikaw, K.-I.; Frank, J.; Christodoulou, D. M.; Koide, S.; Sakai, J.-I.; Sol, H.; Mutel, R. L.

    1998-12-01

    We present 3-D numerical simulations of moderately hot, supersonic jets propagating initially along or obliquely to the field lines of a denser magnetized background medium with Lorentz factors of W=4.56 and evolving in a four-dimensional spacetime. The new results are understood as follows: Relativistic simulations have consistently shown that these jets are effectively heavy and so they do not suffer substantial momentum losses and are not decelerated as efficiently as their nonrelativistic counterparts. In addition, the ambient magnetic field, however strong, can be pushed aside with relative ease by the beam, provided that the degrees of freedom associated with all three spatial dimensions are followed self-consistently in the simulations. This effect is analogous to pushing Japanese ``noren'' or vertical Venetian blinds out of the way while the slats are allowed to bend in 3-D space rather than as a 2-D slab structure. We also simulate jets with the more realistic initial conditions for injecting jets for helical mangetic field, perturbed density, velocity, and internal energy, which are supposed to be caused in the process of jet generation. Three possible explanations for the observed variability are (i) tidal disruption of a star falling into the black hole, (ii) instabilities in the relativistic accretion disk, and (iii) jet-related PRocesses. New results will be reported at the meeting.

  2. The dimension added by 3D scanning and 3D printing of meteorites

    NASA Astrophysics Data System (ADS)

    de Vet, S. J.

    2016-01-01

    An overview for the 3D photodocumentation of meteorites is presented, focussing on two 3D scanning methods in relation to 3D printing. The 3D photodocumention of meteorites provides new ways for the digital preservation of culturally, historically or scientifically unique meteorites. It has the potential for becoming a new documentation standard of meteorites that can exist complementary to traditional photographic documentation. Notable applications include (i.) use of physical properties in dark flight-, strewn field-, or aerodynamic modelling; (ii.) collection research of meteorites curated by different museum collections, and (iii.) public dissemination of meteorite models as a resource for educational users. The possible applications provided by the additional dimension of 3D illustrate the benefits for the meteoritics community.

  3. Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

    PubMed

    Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

    2012-05-01

    The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. PMID:22509834

  4. Evaluation of 3D radio-frequency electromagnetic fields for any matching and coupling conditions by the use of basis functions

    NASA Astrophysics Data System (ADS)

    Tiberi, Gianluigi; Fontana, Nunzia; Monorchio, Agostino; Stara, Riccardo; Retico, Alessandra; Tosetti, Michela

    2015-12-01

    A procedure for evaluating radio-frequency electromagnetic fields in anatomical human models for any matching and coupling conditions is introduced. The procedure resorts to the extraction of basis functions: such basis functions, which represent the fields produced by each individual port without any residual coupling, are derived through an algebraic procedure which uses the S parameter matrix and the fields calculated in one (only) full-wave simulation. The basis functions are then used as building-blocks for calculating the fields for any other S parameter matrix. The proposed approach can be used both for volume coil driven in quadrature and for parallel transmission configuration.

  5. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts.

    PubMed

    Pandey, Sachchida Nand; Khawaja, Hunain; Chen, Yi-Wen

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells. PMID:26007167

  6. Efficient synthesis of spironaphthopyrano [2,3-d]pyrimidine-5,3'-indolines under solvent-free conditions catalyzed by SBA-Pr-SO3H as a nanoporous acid catalyst.

    PubMed

    Ziarani, Ghodsi Mohammadi; Lashgari, Negar; Faramarzi, Sakineh; Badiei, Alireza

    2014-01-01

    A green, simple one-pot synthesis of spironaphthopyrano[2,3-d]pyrimidine-5,3'-indoline derivatives by a three-component reaction of isatins, 2-naphthol, and barbituric acids under solvent-free conditions in the presence of SBA-Pr-SO(3)H has been accomplished. Sulfonic acid functionalized SBA-15 (SBA-Pr-SO(3)H) as a heterogeneous nanoporous solid acid catalyst was found to be an efficient and recyclable acid catalyst in this synthesis. PMID:25286212

  7. Effect of culture conditions on morphological changes of Helicobacter pylori.

    PubMed

    Tominaga, K; Hamasaki, N; Watanabe, T; Uchida, T; Fujiwara, Y; Takaishi, O; Higuchi, K; Arakawa, T; Ishii, E; Kobayashi, K; Yano, I; Kuroki, T

    1999-01-01

    The morphological conversion of Helicobacter pylori from the spiral form to the coccoid form may be the expression of a transitory adaptation to an unsuitable environment. The mechanism(s) of this conversion are not clear. In this study, we examined whether the morphological conversion of H. pylori is affected by various culture conditions, such as oxygen concentration, pH, temperature, or the presence of beta-cyclodextrin. H. pylori (NTCC11916) was cultured on Brucella agar, followed by culture in Brucella broth containing 1% agar under several conditions. Morphological conversion of individual H. pylori on the agar plate was investigated with time after incubation under phase contrast microscopy. When H. pylori was inoculated in Brucella broth containing beta-cyclodextrin, the spiral form of the organism was observed even after 6 days of incubation under standard culture conditions: 37 degrees C, pH 7, and microaerobic atmosphere (5% O2/10% CO2/85% N2) (control). The morphological conversion of H. pylori was completed on day 3 in an aerobic atmosphere (20% O2 supply) and on day 2 in an undermicroaerobic atmosphere (<0.1% O2). Its complete morphological conversion was observed at pH 8 on day 5 and at pH 4 on day 6. All of the H. pylori (100%) incubated at 20 degrees or 42 degrees C had converted from the bacillary to the coccoid form on day 4. Conditioned medium without beta-cyclodextrin caused complete conversion on day 5. These results suggest that oxygen concentration, pH, temperature, and beta-cyclodextrin may be related to the H. pylori morphological conversion from the bacillary to the coccoid form. PMID:10616762

  8. Carotenoid Production by Halophilic Archaea Under Different Culture Conditions.

    PubMed

    Calegari-Santos, Rossana; Diogo, Ricardo Alexandre; Fontana, José Domingos; Bonfim, Tania Maria Bordin

    2016-05-01

    Carotenoids are pigments that may be used as colorants and antioxidants in food, pharmaceutical, and cosmetic industries. Since they also benefit human health, great efforts have been undertaken to search for natural sources of carotenoids, including microbial ones. The optimization of culture conditions to increase carotenoid yield is one of the strategies used to minimize the high cost of carotenoid production by microorganisms. Halophilic archaea are capable of producing carotenoids according to culture conditions. Their main carotenoid is bacterioruberin with 50 carbon atoms. In fact, the carotenoid has important biological functions since it acts as cell membrane reinforcement and it protects the microorganism against DNA damaging agents. Moreover, carotenoid extracts from halophilic archaea have shown high antioxidant capacity. Therefore, current review summarizes the effect of different culture conditions such as salt and carbon source concentrations in the medium, light incidence, and oxygen tension on carotenoid production by halophilic archaea and the strategies such as optimization methodology and two-stage cultivation already used to increase the carotenoid yield of these microorganisms. PMID:26750123

  9. Robust bioengineered 3D functional human intestinal epithelium

    PubMed Central

    Chen, Ying; Lin, Yinan; Davis, Kimberly M.; Wang, Qianrui; Rnjak-Kovacina, Jelena; Li, Chunmei; Isberg, Ralph R.; Kumamoto, Carol A.; Mecsas, Joan; Kaplan, David L.

    2015-01-01

    Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments. PMID:26374193

  10. Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

    USGS Publications Warehouse

    Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Shively, Dawn A.; Nevers, Meredith B.

    2010-01-01

    Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P R = 0.565, P N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R2 = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R2 = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results

  11. A 3D alcoholic liver disease model on a chip.

    PubMed

    Lee, JaeSeo; Choi, BongHwan; No, Da Yoon; Lee, GeonHui; Lee, Seung-Ri; Oh, HyunJik; Lee, Sang-Hoon

    2016-03-14

    Alcohol is one of the main causes of liver diseases, and the development of alcoholic liver disease (ALD) treatment methods has been one of the hottest issues. For this purpose, development of in vitro models mimicking the in vivo physiology is one of the critical requirements, and they help to determine the disease mechanisms and to discover the treatment method. Herein, a three-dimensional (3D) ALD model was developed and its superior features in mimicking the in vivo condition were demonstrated. A spheroid-based microfluidic chip was employed for the development of the 3D in vitro model of ALD progression. We co-cultured rat primary hepatocytes and hepatic stellate cells (HSCs) in a fluidic chip to investigate the role of HSCs in the recovery of liver with ALD. An interstitial level of flow derived by an osmotic pump was applied to the chip to provide in vivo mimicking of fluid activity. Using this in vitro tool, we were able to observe structural changes and decreased hepatic functions with the increase in ethanol concentration. The recovery process of liver injured by alcohol was observed by providing fresh culture medium to the damaged 3D liver tissue for few days. A reversibly- and irreversibly-injured ALD model was established. The proposed model can not only be used for the research of alcoholic disease mechanism, but also has the potential for use in studies of hepatotoxicity and drug screening applications. PMID:26857817

  12. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  13. Grape Cultivar and Sap Culture Conditions Affect the Development of Xylella fastidiosa Phenotypes Associated with Pierce's Disease.

    PubMed

    Hao, Lingyun; Zaini, Paulo A; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

    2016-01-01

    Xylella fastidiosa is a xylem-limited bacterium in plant hosts and causes Pierce's disease (PD) of grapevines, which differ in susceptibility according to the Vitis species (spp.). In this work we compared X. fastidiosa biofilm formation and population dynamics when cultured in xylem saps from PD-susceptible and -resistant Vitis spp. under different conditions. Behaviors in a closed-culture system were compared to those in different sap-renewal cultures that would more closely mimic the physicochemical environment encountered in planta. Significant differences in biofilm formation and growth in saps from PD-susceptible and -resistant spp. were only observed using sap renewal culture. Compared to saps from susceptible V. vinifera, those from PD-resistant V. aestivalis supported lower titers of X. fastidiosa and less biofilm and V. champinii suppressed both growth and biofilm formation, behaviors which are correlated with disease susceptibility. Furthermore, in microfluidic chambers X. fastidiosa formed thick mature biofilm with three-dimensional (3-D) structures, such as pillars and mounds, in saps from all susceptible spp. In contrast, only small aggregates of various shapes were formed in saps from four out of five of the resistant spp.; sap from the resistant spp. V. mustangensis was an exception in that it also supported thick lawns of biofilm but not the above described 3-D structures typically seen in a mature biofilm from the susceptible saps. Our findings provide not only critical technical information for future bioassays, but also suggest further understanding of PD susceptibility. PMID:27508296

  14. Grape Cultivar and Sap Culture Conditions Affect the Development of Xylella fastidiosa Phenotypes Associated with Pierce's Disease

    PubMed Central

    Hoch, Harvey C.; Burr, Thomas J.; Mowery, Patricia

    2016-01-01

    Xylella fastidiosa is a xylem-limited bacterium in plant hosts and causes Pierce’s disease (PD) of grapevines, which differ in susceptibility according to the Vitis species (spp.). In this work we compared X. fastidiosa biofilm formation and population dynamics when cultured in xylem saps from PD-susceptible and -resistant Vitis spp. under different conditions. Behaviors in a closed-culture system were compared to those in different sap-renewal cultures that would more closely mimic the physicochemical environment encountered in planta. Significant differences in biofilm formation and growth in saps from PD-susceptible and -resistant spp. were only observed using sap renewal culture. Compared to saps from susceptible V. vinifera, those from PD-resistant V. aestivalis supported lower titers of X. fastidiosa and less biofilm and V. champinii suppressed both growth and biofilm formation, behaviors which are correlated with disease susceptibility. Furthermore, in microfluidic chambers X. fastidiosa formed thick mature biofilm with three-dimensional (3-D) structures, such as pillars and mounds, in saps from all susceptible spp. In contrast, only small aggregates of various shapes were formed in saps from four out of five of the resistant spp.; sap from the resistant spp. V. mustangensis was an exception in that it also supported thick lawns of biofilm but not the above described 3-D structures typically seen in a mature biofilm from the susceptible saps. Our findings provide not only critical technical information for future bioassays, but also suggest further understanding of PD susceptibility. PMID:27508296

  15. 3D Spectroscopy in Astronomy

    NASA Astrophysics Data System (ADS)

    Mediavilla, Evencio; Arribas, Santiago; Roth, Martin; Cepa-Nogué, Jordi; Sánchez, Francisco

    2011-09-01

    Preface; Acknowledgements; 1. Introductory review and technical approaches Martin M. Roth; 2. Observational procedures and data reduction James E. H. Turner; 3. 3D Spectroscopy instrumentation M. A. Bershady; 4. Analysis of 3D data Pierre Ferruit; 5. Science motivation for IFS and galactic studies F. Eisenhauer; 6. Extragalactic studies and future IFS science Luis Colina; 7. Tutorials: how to handle 3D spectroscopy data Sebastian F. Sánchez, Begona García-Lorenzo and Arlette Pécontal-Rousset.

  16. 3D Elevation Program—Virtual USA in 3D

    USGS Publications Warehouse

    Lukas, Vicki; Stoker, J.M.

    2016-01-01

    The U.S. Geological Survey (USGS) 3D Elevation Program (3DEP) uses a laser system called ‘lidar’ (light detection and ranging) to create a virtual reality map of the Nation that is very accurate. 3D maps have many uses with new uses being discovered all the time.  

  17. A 3D radiative transfer framework. VI. PHOENIX/3D example applications

    NASA Astrophysics Data System (ADS)

    Hauschildt, P. H.; Baron, E.

    2010-01-01

    Aims: We demonstrate the application of our 3D radiative transfer framework in the model atmosphere code PHOENIX for a number of spectrum synthesis calculations for very different conditions. Methods: The 3DRT framework discussed in the previous papers of this series was added to our general-purpose model atmosphere code PHOENIX/1D and an extended 3D version PHOENIX/3D was created. The PHOENIX/3D code is parallelized via the MPI library using a hierarchical domain decomposition and displays very good strong scaling. Results: We present the results of several test cases for widely different atmosphere conditions and compare the 3D calculations with equivalent 1D models to assess the internal accuracy of the 3D modeling. In addition, we show the results for a number of parameterized 3D structures. Conclusions: With presently available computational resources it is possible to solve the full 3D radiative transfer (including scattering) problem with the same micro-physics as included in 1D modeling.

  18. Assessing 3d Photogrammetry Techniques in Craniometrics

    NASA Astrophysics Data System (ADS)

    Moshobane, M. C.; de Bruyn, P. J. N.; Bester, M. N.

    2016-06-01

    Morphometrics (the measurement of morphological features) has been revolutionized by the creation of new techniques to study how organismal shape co-varies with several factors such as ecophenotypy. Ecophenotypy refers to the divergence of phenotypes due to developmental changes induced by local environmental conditions, producing distinct ecophenotypes. None of the techniques hitherto utilized could explicitly address organismal shape in a complete biological form, i.e. three-dimensionally. This study investigates the use of the commercial software, Photomodeler Scanner® (PMSc®) three-dimensional (3D) modelling software to produce accurate and high-resolution 3D models. Henceforth, the modelling of Subantarctic fur seal (Arctocephalus tropicalis) and Antarctic fur seal (Arctocephalus gazella) skulls which could allow for 3D measurements. Using this method, sixteen accurate 3D skull models were produced and five metrics were determined. The 3D linear measurements were compared to measurements taken manually with a digital caliper. In addition, repetitive measurements were recorded by varying researchers to determine repeatability. To allow for comparison straight line measurements were taken with the software, assuming that close accord with all manually measured features would illustrate the model's accurate replication of reality. Measurements were not significantly different demonstrating that realistic 3D skull models can be successfully produced to provide a consistent basis for craniometrics, with the additional benefit of allowing non-linear measurements if required.

  19. CFL3D, FUN3d, and NSU3D Contributions to the Fifth Drag Prediction Workshop

    NASA Technical Reports Server (NTRS)

    Park, Michael A.; Laflin, Kelly R.; Chaffin, Mark S.; Powell, Nicholas; Levy, David W.

    2013-01-01

    Results presented at the Fifth Drag Prediction Workshop using CFL3D, FUN3D, and NSU3D are described. These are calculations on the workshop provided grids and drag adapted grids. The NSU3D results have been updated to reflect an improvement to skin friction calculation on skewed grids. FUN3D results generated after the workshop are included for custom participant generated grids and a grid from a previous workshop. Uniform grid refinement at the design condition shows a tight grouping in calculated drag, where the variation in the pressure component of drag is larger than the skin friction component. At this design condition, A fine-grid drag value was predicted with a smaller drag adjoint adapted grid via tetrahedral adaption to a metric and mixed-element subdivision. The buffet study produced larger variation than the design case, which is attributed to large differences in the predicted side-of-body separation extent. Various modeling and discretization approaches had a strong impact on predicted side-of-body separation. This large wing root separation bubble was not observed in wind tunnel tests indicating that more work is necessary in modeling wing root juncture flows to predict experiments.

  20. User experience while viewing stereoscopic 3D television

    PubMed Central

    Read, Jenny C.A.; Bohr, Iwo

    2014-01-01

    3D display technologies have been linked to visual discomfort and fatigue. In a lab-based study with a between-subjects design, 433 viewers aged from 4 to 82 years watched the same movie in either 2D or stereo 3D (S3D), and subjectively reported on a range of aspects of their viewing experience. Our results suggest that a minority of viewers, around 14%, experience adverse effects due to viewing S3D, mainly headache and eyestrain. A control experiment where participants viewed 2D content through 3D glasses suggests that around 8% may report adverse effects which are not due directly to viewing S3D, but instead are due to the glasses or to negative preconceptions about S3D (the ‘nocebo effect'). Women were slightly more likely than men to report adverse effects with S3D. We could not detect any link between pre-existing eye conditions or low stereoacuity and the likelihood of experiencing adverse effects with S3D. Practitioner Summary: Stereoscopic 3D (S3D) has been linked to visual discomfort and fatigue. Viewers watched the same movie in either 2D or stereo 3D (between-subjects design). Around 14% reported effects such as headache and eyestrain linked to S3D itself, while 8% report adverse effects attributable to 3D glasses or negative expectations. PMID:24874550

  1. Modular 3-D Transport model

    EPA Science Inventory

    MT3D was first developed by Chunmiao Zheng in 1990 at S.S. Papadopulos & Associates, Inc. with partial support from the U.S. Environmental Protection Agency (USEPA). Starting in 1990, MT3D was released as a pubic domain code from the USEPA. Commercial versions with enhanced capab...

  2. Market study: 3-D eyetracker

    NASA Technical Reports Server (NTRS)

    1977-01-01

    A market study of a proposed version of a 3-D eyetracker for initial use at NASA's Ames Research Center was made. The commercialization potential of a simplified, less expensive 3-D eyetracker was ascertained. Primary focus on present and potential users of eyetrackers, as well as present and potential manufacturers has provided an effective means of analyzing the prospects for commercialization.

  3. LLNL-Earth3D

    2013-10-01

    Earth3D is a computer code designed to allow fast calculation of seismic rays and travel times through a 3D model of the Earth. LLNL is using this for earthquake location and global tomography efforts and such codes are of great interest to the Earth Science community.

  4. [3-D ultrasound in gastroenterology].

    PubMed

    Zoller, W G; Liess, H

    1994-06-01

    Three-dimensional (3D) sonography represents a development of noninvasive diagnostic imaging by real-time two-dimensional (2D) sonography. The use of transparent rotating scans, comparable to a block of glass, generates a 3D effect. The objective of the present study was to optimate 3D presentation of abdominal findings. Additional investigations were made with a new volumetric program to determine the volume of selected findings of the liver. The results were compared with the estimated volumes of 2D sonography and 2D computer tomography (CT). For the processing of 3D images, typical parameter constellations were found for the different findings, which facilitated processing of 3D images. In more than 75% of the cases examined we found an optimal 3D presentation of sonographic findings with respect to the evaluation criteria developed by us for the 3D imaging of processed data. Great differences were found for the estimated volumes of the findings of the liver concerning the three different techniques applied. 3D ultrasound represents a valuable method to judge morphological appearance in abdominal findings. The possibility of volumetric measurements enlarges its potential diagnostic significance. Further clinical investigations are necessary to find out if definite differentiation between benign and malign findings is possible. PMID:7919882

  5. 3D World Building System

    SciTech Connect

    2013-10-30

    This video provides an overview of the Sandia National Laboratories developed 3-D World Model Building capability that provides users with an immersive, texture rich 3-D model of their environment in minutes using a laptop and color and depth camera.

  6. 3D World Building System

    ScienceCinema

    None

    2014-02-26

    This video provides an overview of the Sandia National Laboratories developed 3-D World Model Building capability that provides users with an immersive, texture rich 3-D model of their environment in minutes using a laptop and color and depth camera.

  7. Euro3D Science Conference

    NASA Astrophysics Data System (ADS)

    Walsh, J. R.

    2004-02-01

    The Euro3D RTN is an EU funded Research Training Network to foster the exploitation of 3D spectroscopy in Europe. 3D spectroscopy is a general term for spectroscopy of an area of the sky and derives its name from its two spatial + one spectral dimensions. There are an increasing number of instruments which use integral field devices to achieve spectroscopy of an area of the sky, either using lens arrays, optical fibres or image slicers, to pack spectra of multiple pixels on the sky (``spaxels'') onto a 2D detector. On account of the large volume of data and the special methods required to reduce and analyse 3D data, there are only a few centres of expertise and these are mostly involved with instrument developments. There is a perceived lack of expertise in 3D spectroscopy spread though the astronomical community and its use in the armoury of the observational astronomer is viewed as being highly specialised. For precisely this reason the Euro3D RTN was proposed to train young researchers in this area and develop user tools to widen the experience with this particular type of data in Europe. The Euro3D RTN is coordinated by Martin M. Roth (Astrophysikalisches Institut Potsdam) and has been running since July 2002. The first Euro3D science conference was held in Cambridge, UK from 22 to 23 May 2003. The main emphasis of the conference was, in keeping with the RTN, to expose the work of the young post-docs who are funded by the RTN. In addition the team members from the eleven European institutes involved in Euro3D also presented instrumental and observational developments. The conference was organized by Andy Bunker and held at the Institute of Astronomy. There were over thirty participants and 26 talks covered the whole range of application of 3D techniques. The science ranged from Galactic planetary nebulae and globular clusters to kinematics of nearby galaxies out to objects at high redshift. Several talks were devoted to reporting recent observations with newly

  8. 3-D Finite Element Heat Transfer

    1992-02-01

    TOPAZ3D is a three-dimensional implicit finite element computer code for heat transfer analysis. TOPAZ3D can be used to solve for the steady-state or transient temperature field on three-dimensional geometries. Material properties may be temperature-dependent and either isotropic or orthotropic. A variety of time-dependent and temperature-dependent boundary conditions can be specified including temperature, flux, convection, and radiation. By implementing the user subroutine feature, users can model chemical reaction kinetics and allow for any type of functionalmore » representation of boundary conditions and internal heat generation. TOPAZ3D can solve problems of diffuse and specular band radiation in an enclosure coupled with conduction in the material surrounding the enclosure. Additional features include thermal contact resistance across an interface, bulk fluids, phase change, and energy balances.« less

  9. PLOT3D user's manual

    NASA Technical Reports Server (NTRS)

    Walatka, Pamela P.; Buning, Pieter G.; Pierce, Larry; Elson, Patricia A.

    1990-01-01

    PLOT3D is a computer graphics program designed to visualize the grids and solutions of computational fluid dynamics. Seventy-four functions are available. Versions are available for many systems. PLOT3D can handle multiple grids with a million or more grid points, and can produce varieties of model renderings, such as wireframe or flat shaded. Output from PLOT3D can be used in animation programs. The first part of this manual is a tutorial that takes the reader, keystroke by keystroke, through a PLOT3D session. The second part of the manual contains reference chapters, including the helpfile, data file formats, advice on changing PLOT3D, and sample command files.

  10. 3D printing in dentistry.

    PubMed

    Dawood, A; Marti Marti, B; Sauret-Jackson, V; Darwood, A

    2015-12-01

    3D printing has been hailed as a disruptive technology which will change manufacturing. Used in aerospace, defence, art and design, 3D printing is becoming a subject of great interest in surgery. The technology has a particular resonance with dentistry, and with advances in 3D imaging and modelling technologies such as cone beam computed tomography and intraoral scanning, and with the relatively long history of the use of CAD CAM technologies in dentistry, it will become of increasing importance. Uses of 3D printing include the production of drill guides for dental implants, the production of physical models for prosthodontics, orthodontics and surgery, the manufacture of dental, craniomaxillofacial and orthopaedic implants, and the fabrication of copings and frameworks for implant and dental restorations. This paper reviews the types of 3D printing technologies available and their various applications in dentistry and in maxillofacial surgery. PMID:26657435

  11. Cultural conditionally and aid to education in east Africa

    NASA Astrophysics Data System (ADS)

    Brock-Utne, Birgit

    1995-05-01

    Aid to African education often involves the imposition of conditions that create dependency and undermine indigenous educational patterns. Such conditions can include the insistence on textbooks written and published abroad, the use of examination systems devised in Europe or North America, and the neglect of African culture and languages. In the first part of this article the author examines the attempts at educational reform after the African states had achieved independence. The second part highlights the renewed dangers of educational dependency following the 1990 World Conference on Education for All, held in Jomtien, Thailand. The last part of the article discusses whether there is such a thing as "educational aid for empowerment" and gives some examples of good educational aid projects in Africa.

  12. Scoops3D: software to analyze 3D slope stability throughout a digital landscape

    USGS Publications Warehouse

    Reid, Mark E.; Christian, Sarah B.; Brien, Dianne L.; Henderson, Scott T.

    2015-01-01

    The computer program, Scoops3D, evaluates slope stability throughout a digital landscape represented by a digital elevation model (DEM). The program uses a three-dimensional (3D) method of columns approach to assess the stability of many (typically millions) potential landslides within a user-defined size range. For each potential landslide (or failure), Scoops3D assesses the stability of a rotational, spherical slip surface encompassing many DEM cells using a 3D version of either Bishop’s simplified method or the Ordinary (Fellenius) method of limit-equilibrium analysis. Scoops3D has several options for the user to systematically and efficiently search throughout an entire DEM, thereby incorporating the effects of complex surface topography. In a thorough search, each DEM cell is included in multiple potential failures, and Scoops3D records the lowest stability (factor of safety) for each DEM cell, as well as the size (volume or area) associated with each of these potential landslides. It also determines the least-stable potential failure for the entire DEM. The user has a variety of options for building a 3D domain, including layers or full 3D distributions of strength and pore-water pressures, simplistic earthquake loading, and unsaturated suction conditions. Results from Scoops3D can be readily incorporated into a geographic information system (GIS) or other visualization software. This manual includes information on the theoretical basis for the slope-stability analysis, requirements for constructing and searching a 3D domain, a detailed operational guide (including step-by-step instructions for using the graphical user interface [GUI] software, Scoops3D-i) and input/output file specifications, practical considerations for conducting an analysis, results of verification tests, and multiple examples illustrating the capabilities of Scoops3D. Easy-to-use software installation packages are available for the Windows or Macintosh operating systems; these packages

  13. PLOT3D/AMES, APOLLO UNIX VERSION USING GMR3D (WITH TURB3D)

    NASA Technical Reports Server (NTRS)

    Buning, P.

    1994-01-01

    PLOT3D is an interactive graphics program designed to help scientists visualize computational fluid dynamics (CFD) grids and solutions. Today, supercomputers and CFD algorithms can provide scientists with simulations of such highly complex phenomena that obtaining an understandi