WorldWideScience

Sample records for 3d cell cultures

  1. 3D Cell Culture in Alginate Hydrogels

    Therese Andersen

    2015-03-01

    Full Text Available 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.

  2. Multizone paper platform for 3D cell cultures.

    Ratmir Derda

    Full Text Available 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.

  3. Fabrication of Nanostructured Poly-ε-caprolactone 3D Scaffolds for 3D Cell Culture Technology

    Schipani, Rossana

    2015-04-21

    Tissue engineering is receiving tremendous attention due to the necessity to overcome the limitations related to injured or diseased tissues or organs. It is the perfect combination of cells and biomimetic-engineered materials. With the appropriate biochemical factors, it is possible to develop new effective bio-devices that are capable to improve or replace biological functions. Latest developments in microfabrication methods, employing mostly synthetic biomaterials, allow the production of three-dimensional (3D) scaffolds that are able to direct cell-to-cell interactions and specific cellular functions in order to drive tissue regeneration or cell transplantation. The presented work offers a rapid and efficient method of 3D scaffolds fabrication by using optical lithography and micro-molding techniques. Bioresorbable polymer poly-ε-caprolactone (PCL) was the material used thanks to its high biocompatibility and ability to naturally degrade in tissues. 3D PCL substrates show a particular combination in the designed length scale: cylindrical shaped pillars with 10μm diameter, 10μm height, arranged in a hexagonal lattice with spacing of 20μm were obtained. The sidewalls of the pillars were nanostructured by attributing a 3D architecture to the scaffold. The suitability of these devices as cell culture technology supports was evaluated by plating NIH/3T3 mouse embryonic fibroblasts and human Neural Stem Cells (hNSC) on them. Scanning Electron Microscopy (SEM) analysis was carried out in order to examine the micro- and nano-patterns on the surface of the supports. In addition, after seeding of cells, SEM and immunofluorescence characterization of the fabricated systems were performed to check adhesion, growth and proliferation. It was observed that cells grow and develop healthy on the bio-polymeric devices by giving rise to well-interconnected networks. 3D PCL nano-patterned pillared scaffold therefore may have considerable potential as effective tool for

  4. Surface modified alginate microcapsules for 3D cell culture

    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.

  5. Self-Assembled Peptide Gels for 3D Cell Culture

    Tang, Claire

    2010-01-01

    Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progre...

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

    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.

  7. Molecular predictors of 3D morphogenesis by breast cancer cell lines in 3D culture.

    Ju Han

    2010-02-01

    Full Text Available 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, PPARgamma has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARgamma has been validated through two supporting biological assays.

  8. Impedance Spectroscopic Characterisation of Porosity in 3D Cell Culture Scaffolds with Different Channel Networks

    Canali, Chiara; Mohanty, Soumyaranjan; Heiskanen, Arto;

    2015-01-01

    We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity...... serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures....

  9. Development of 3-D Hydrogel Culture Systems With On-Demand Cell Separation

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

    2013-01-01

    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 3-D co-culture methods lack the ability to effectively separate 2 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 3-D hydrogel co-culture system...

  10. Design of 3D printed insert for hanging culture of Caco-2 cells

    A Caco-2 cell culture on Transwell, an alternative testing to animal or human testing used in evaluating drug intestinal permeability, incorrectly estimated the absorption of actively transported drugs due to the low expression of membrane transporters. Similarly, three-dimensional (3D) cultures of Caco-2 cells, which have been recommended to be more physiological relevant, were not superior to the Transwell culture in either accuracy or convenience in drug permeability testing. Using rapid 3D printing prototyping techniques, this study proposed a hanging culture of Caco-2 cells that performed with high accuracy in predicting drug permeability in humans. As found, hanging cultured Caco-2 cells formed a confluent monolayer and maintained high cell viability on the 3D printed insert. Compared with the normal culture on Transwell, the Caco-2 cells on the 3D printed insert presented ∼30–100% higher brush border enzyme activity and ∼2–7 folds higher activity of P-glycoprotein/multidrug resistance-associated protein 2 during 21 days of incubation. For the eight membrane transporter substrates, the predictive curve of the 3D printing culture exhibited better linearity (R2 = 0.92) to the human oral adsorption than that of the Transwell culture (R2 = 0.84), indicating better prediction by the 3D printing culture. In this regard, the 3D printed insert for hanging culture could be potentially developed as a convenient and low-cost tool for testing drug oral absorption. (paper)

  11. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

    Hanley Edward N

    2000-10-01

    Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

  12. Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture

    Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

  13. Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture

    Frampton, J P; Hynd, M R; Shain, W [Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12210 (United States); Shuler, M L, E-mail: jf7674@albany.edu [Department of Biomedical Engineering, 270 Olin Hall, Cornell University, Ithaca, NY 14850 (United States)

    2011-02-15

    Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

  14. Bioimpedance monitoring of 3D cell culturing-Complementary electrode configurations for enhanced spatial sensitivity

    Canali, Chiara; Heiskanen, Arto; Muhammad, Haseena Bashir;

    2015-01-01

    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...... 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...... 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...

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

    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

  16. Gel de plaquetas: arcabouço 3D para cultura celular Platelet gel: 3D scaffold for cell culture

    Andrei Moroz

    2009-01-01

    Full Text Available INTRODUÇÃO: O reparo tissular é o objetivo final da cirurgia. A cultura celular requer arcabouço mecânico que dê suporte ao crescimento celular e difusão dos nutrientes. O uso do plasma rico em plaquetas (PRP como um arcabouço 3D possui diversas vantagens: é material biológico, de fácil absorção pós-transplante, rico em fatores de crescimento, em especial PDGF- ββ e TGF-β que estimula síntese de matriz extracelular na cartilagem. OBJETIVO: Desenvolver arcabouço 3D à base de PRP. MATERIAIS E MÉTODOS: Duas formas foram idealizadas: Sphere e Carpet. Condições estéreis foram utilizadas. O gel de plaquetas permaneceu em cultura celular, observado diariamente em microscópio invertido. RESULTADOS: Ambos arcabouços obtiveram sucesso, com aspectos positivos e negativos. DISCUSSÃO: A forma Sphere não aderiu ao plástico. Observou-se retração do gel e investigação ao microscópio dificultada devido às áreas opacas no campo visual. A forma Carpet não aderiu ao plástico e apresentou-se translúcida. O tempo de estudo foi de 20 dias. CONCLUSÕES: A produção de um arcabouço 3D PRP foi um sucesso, e trata-se de uma alternativa que necessita ser mais utilizado e investigado para que se consolide em uma rota eficiente e confiável na tecnologia de engenharia tissular, particularmente em cultura de tecido cartilaginoso.INTRODUCTION: Tissue repair has been the ultimate goal of surgery. Cell culture requires a mechanical scaffold that supports cell growth and nutrient diffusion. Using platelet-rich plasma (PRP as a 3D scaffold presents various advantages: it is a biological material, easily absorbed after transplantation, rich in growth factors, in particular, PDGF-ββ and TGF-β that stimulate extracellular matrix synthesis in cartilage culture. OBJECTIVE: To develop a PRP 3D scaffold. Material and METHODS: Two forms were idealized: Sphere and Carpet. Sterile conditions were used. The platelet gel remained in culture

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

    Akpe Victor; Rydholm Susanna; Liebmann Thomas; Brismar Hjalmar

    2007-01-01

    Abstract 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-derive...

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

    Pranav Joshi

    2015-12-01

    Full Text Available 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.

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

    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

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

    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

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

    Akpe Victor

    2007-12-01

    Full Text Available Abstract 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.

  2. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

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

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

    2011-04-01

    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 microfluidic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact. PMID:21113663

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

    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

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

    Cemal Cagatay Bilgin

    Full Text Available 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.

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

    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

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

    Paun, Irina Alexandra, E-mail: irina.paun@physics.pub.ro [National Institute for Laser, Plasma and Radiation Physics, RO-077125, Magurele, Bucharest (Romania); Faculty of Applied Sciences, University Politehnica of Bucharest, RO-060042, Bucharest (Romania); Mihailescu, Mona [Faculty of Applied Sciences, University Politehnica of Bucharest, RO-060042, Bucharest (Romania); Calenic, Bogdan [Department of Biochemistry, Faculty of Dentistry, UMF Carol Davila, Bucharest (Romania); Luculescu, Catalin Romeo [National Institute for Laser, Plasma and Radiation Physics, RO-077125, Magurele, Bucharest (Romania); Greabu, Maria [Department of Biochemistry, Faculty of Dentistry, UMF Carol Davila, Bucharest (Romania); Dinescu, Maria, E-mail: dinescum@nipne.ro [National Institute for Laser, Plasma and Radiation Physics, RO-077125, Magurele, Bucharest (Romania)

    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 μm{sup 2} 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.

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

    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.

  9. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  10. 3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

    Kim, Yeonhee; Rajagopalan, Padmavathy

    2010-01-01

    Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and

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

    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.

  12. Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

    M. Riccio; Resca, E.; Maraldi, T; Pisciotta, A.; Ferrari, A; Bruzzesi, G.; De Pol, A.

    2010-01-01

    The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurr...

  13. Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

    M. Riccio

    2010-11-01

    Full Text Available The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, MatrigelTM and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in MatrigelTM DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.

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

    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

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

    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

  16. 3D cell culture systems modeling tumor growth determinants in cancer target discovery.

    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

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

    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

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

    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

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

    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 t...

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

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

    2010-01-01

    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 t...

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

    Chitcholtan, Kenny, E-mail: kenny.chitcholtan@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Asselin, Eric, E-mail: Eric.Asselin@uqtr.ca [Department of Chemistry and Biology, University of Quebec, at Trois-Rivières, C.P. 500, Trois-Rivières, Quebec, Canada G9A 5H7 (Canada); Parent, Sophie, E-mail: Sophie.Parent@uqtr.ca [Department of Chemistry and Biology, University of Quebec, at Trois-Rivières, C.P. 500, Trois-Rivières, Quebec, Canada G9A 5H7 (Canada); Sykes, Peter H., E-mail: peter.sykes@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Evans, John J., E-mail: john.evans@otago.ac.nz [Department of Obstetrics and Gynaecology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand); Centre of Neuroendocrinology and The MacDiarmid Institute of Advanced Materials and Nanotechnology, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8011 (New Zealand)

    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.

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

    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 E2 (PGE2) 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.

  3. A Novel Flow-Perfusion Bioreactor Supports 3D Dynamic Cell Culture

    Alexander M. Sailon

    2009-01-01

    Full Text Available Background. Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm. A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm scaffolds. Methods. Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. Results. By day 8, static scaffolds had a periphery cell density of 67%±5.0%, while in the core it was 0.3%±0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94%±8.3% and core density of 76%±3.1% at day 8. Conclusions. Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

  4. Dual targeting of EGFR and focal adhesion kinase in 3D grown HNSCC cell cultures

    Purpose: Epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) show frequent overexpression and hyperactivity in various human malignancies including head and neck squamous cell carcinomas (HNSCC). To examine effects of dual EGFR/FAK inhibition on cellular radiosensitivity of HNSCC cells in a more physiological environment, we employed a previously established laminin-rich extracellular matrix (lrECM) based three-dimensional (3D) cell culture model. Materials and methods: UTSCC15 and SAS HNSCC cell lines stably transfected with EGFR-CFP or CFP were used. Single or combined EGFR (Cetuximab, siRNA) and FAK (TAE226, siRNA) inhibition were accomplished prior to measuring clonogenic survival and protein expression and phosphorylation. Immunofluorescence enabled visualization of EGFR-CFP and FAK. Results: Cetuximab resulted in higher radiosensitization in EGFR-CFP overexpressing cell lines than CFP controls. Single EGFR or FAK inhibition mediated radiosensitization, while dual EGFR/FAK targeting further augmented this effect. Despite signaling alterations upon Cetuximab and siRNA knockdown, analysis of protein expression and phosphorylation indicates EGFR and FAK signaling coexistence without obvious overlap. Conclusions: Combined EGFR/FAK targeting yielded stronger radiosensitization than either approach alone, which might be based on non-overlapping downstream signaling. Whether dual targeting of EGFR and FAK can reasonably be combined with radiotherapy and chemotherapy needs clarification.

  5. A Multifunctional 3D Co-Culture System for Studies of Mammary Tissue Morphogenesis and Stem Cell Biology

    Campbell, Jonathan J.; Davidenko, Natalia; Caffarel, Maria M.; Cameron, Ruth E.; Watson, Christine J

    2011-01-01

    Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this sc...

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

    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

  7. AlgiMatrix™ Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

    Godugu, Chandraiah; Patel, Apurva R.; Desai, Utkarsh; Andey, Terrick; Sams, Alexandria; Singh, Mandip

    2013-01-01

    Background Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. Methods Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without co...

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

    Tao Wang

    2015-12-01

    Full Text Available 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

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

    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

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

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

    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

  12. The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.

    O'Driscoll, Lorraine

    2016-01-01

    PUBLISHED 2016 Jun 10. doi: 10.18632/oncotarget.9935. [Epub ahead of print] Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing c...

  13. Increased GFAP immunoreactivity by astrocytes in response to contact with dorsal root ganglia cells in a 3D culture model

    East, Emma; Golding, Jon; Phillips, James

    2007-01-01

    Failure of repair mechanisms in the injured CNS is widely attributed to the inhibitory environment of the lesion site, most notably the formation of the glial scar which forms a physical and physiological barrier to axon regeneration. We developed an in vitro 3D cell culture model to investigate the response of astrocytes to cells found at the inhibitory interfaces formed following damage to the spinal cord. CellTrackerTM labelled dissociated DRGs were seeded onto astrocy...

  14. AlgiMatrix™ based 3D cell culture system as an in-vitro tumor model for anticancer studies.

    Chandraiah Godugu

    Full Text Available BACKGROUND: Three-dimensional (3D in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. METHODS: Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100-300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin and nanoparticle (NLC were done using spheroids. RESULTS: IC(50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. CONCLUSION: The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

  15. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Jonathan J Campbell

    Full Text Available Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM in three dimensional (3D space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  16. 3D cell cultures of human head and neck squamous cell carcinoma cells are radiosensitized by the focal adhesion kinase inhibitor TAE226

    Background and purpose: Focal adhesion kinase (FAK), a main player in integrin signaling and survival, is frequently overexpressed in human cancers and therefore postulated as potential target in cancer therapy. The aim of this study was to evaluate the radiosensitizing potential of the FAK inhibitor TAE226 in three-dimensional (3D) tumor cell cultures. Materials and methods: Head and neck squamous cell carcinoma (HNSCC) cells (FaDu, UT-SCC15, UT-SCC45), lung cancer cells (A549), colorectal carcinoma cells (DLD-1, HCT-116) and pancreatic tumor cells (MiaPaCa2, Panc1) were treated with different concentrations of TAE226 (0-1 μm; 1 or 24 h) without or in combination with irradiation (0-6 Gy, X-ray, single dose). Subsequently, 3D clonogenic survival assays (laminin-rich extracellular matrix) and Western blotting (expression/phosphorylation, e.g. FAK, Akt, ERK1/2) were performed. Results: All investigated 3D cell cultures showed a dose-dependent reduction in clonogenic survival by TAE226. Intriguingly, TAE226 only significantly radiosensitized 3D HNSCC cell cultures accompanied by a pronounced dephosphorylation of FAK, Akt and ERK1/2. Conclusions: Our data demonstrate TAE226 as potent FAK inhibitor that enhances the cellular radiosensitivity particularly of HNSCC cells grown in a 3D cell culture model. Future in vitro and in vivo investigations will clarify, to which extent this approach might be clinically relevant for radiotherapy of HNSCC.

  17. The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures

    Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D) in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks), the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to induce near-complete tumor phenotype reversion. These human

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

    Babak Qasemi-Panahi

    2013-02-01

    Full Text Available 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.

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

    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

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

    Ko, Jae Hyung [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Kim, Yang Hee [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Jeong, Seong Hee; Lee, Song [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Park, Si-Nae [Regenerative Medicine Research Center, Dalim Tissen Co., LTD., 383-93, Yonnam-Dong, Mapo-gu, Seoul (Korea, Republic of); Shim, In Kyong, E-mail: shimiink@gmail.com [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Kim, Song Cheol, E-mail: drksc@amc.seoul.kr [Asan Institute for Life Science, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of); Department of Surgery, University of Ulsan College of Medicine & Asan Medical Center, 388-1 Pungnap-2 Dong, Songpa-gu, Seoul (Korea, Republic of)

    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.

  1. Design, construction and testing of a COC 3D flow-over flow-through bioreactor for hepatic cell culture

    Windels, Jindrich; Verplancke, Rik; Jahanshahi, Amir; Heimann, Marcus; Leite, Sofia B.; Roosens, Tiffany; van Grunsven, Leo A; Barbe, Laurent; Prill, Sebastian; Jaeger, Magnus; Duschl, Claus; Vanfleteren, Jan

    2015-01-01

    In this poster, we present the joint development efforts for a 3D microfluidic bioreactor for hepatic cell cultures. Cyclic Olefin Copolymer (COC) was selected for constructing the bioreactor, since the material has good chemical resistance, low adsorption and good optical properties, including low auto-fluorescence. A downside of COC is that it is much more difficult to structure than more traditional microfluidic materials, such as PDMS, PMMA, … Two parallel approaches were developed for...

  2. A photopolymerizable hydrogel for 3-D culture of human embryonic stem cell-derived cardiomyocytes and rat neonatal cardiac cells.

    Shapira-Schweitzer, Keren; Habib, Manhal; Gepstein, Lior; Seliktar, Dror

    2009-02-01

    The purpose of this study was to assess the in vitro ability of two types of cardiomyocytes (cardiomyocytes derived from human embryonic stem cells (hESC-CM) and rat neonatal cardiomyocytes (rN-CM)) to survive and generate a functional cardiac syncytium in a three-dimensional in situ polymerizable hydrogel environment. Each cell type was cultured in a PEGylated fibrinogen (PF) hydrogel for up to two weeks while maturation and cardiac function were documented in terms of spontaneous contractile behavior and biomolecular organization. Quantitative contractile parameters including contraction amplitude and synchronization were measured by non-invasive image analysis. The rN-CM demonstrated the fastest maturation and the most significant spontaneous contraction. The hESC-CM maturation occurred between 10-14 days in culture, and exhibited less contraction amplitude and synchronization in comparison to the rN-CMs. The maturation of both cell types within the hydrogels was confirmed by cardiac-specific biomolecular markers, including alpha-sarcomeric actin, actinin, and connexin-43. Cellular responsiveness to isoproterenol, carbamylcholine and heptanol provided further evidence of the cardiac maturation in the 3-D PF hydrogel as well as identified a potential to use this system for in vitro drug screening. These findings indicate that the PF hydrogel biomaterial can be used as an in situ polymerizable biomaterial for stem cells and their cardiomyocyte derivatives. PMID:19027751

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

    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

  4. 3D PHOTOGRAPHS IN CULTURAL HERITAGE

    Schuhr, W.; J. D. Lee; Kiel, St.

    2013-01-01

    This paper on providing "oo-information" (= objective object-information) on cultural monuments and sites, based on 3D photographs is also a contribution of CIPA task group 3 to the 2013 CIPA Symposium in Strasbourg. To stimulate the interest in 3D photography for scientists as well as for amateurs, 3D-Masterpieces are presented. Exemplary it is shown, due to their high documentary value ("near reality"), 3D photography support, e.g. the recording, the visualization, the interpret...

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

    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.

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

    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

  7. The Effects of GDF-5 and Uniaxial Strain on Mesenchymal Stem Cells in 3-D Culture

    Farng, Eugene; Urdaneta, Alfonso R.; Barba, David; Esmende, Sean; McAllister, David R.

    2008-01-01

    Recent endeavors in tissue engineering have attempted to identify the optimal parameters to create an artificial ligament. Both mechanical and biochemical stimulation have been used by others to independently modulate growth and differentiation, although few studies have explored their interactions. We applied previously described fabrication techniques to create a highly porous (90%–95% porosity, 212–300 μm), 3-D, bioabsorbable polymer scaffold (polycaprolactone). Scaffolds were coated with ...

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

    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

  9. Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

    Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification

  10. Visible light cured thiol-vinyl hydrogels with tunable degradation for 3D cell culture

    Hao, Yiting; Shih, Han; Muňoz, Zachary; Kemp, Arika; Lin, Chien-Chi

    2013-01-01

    We report here a synthetically simple yet highly tunable and diverse visible light mediated thiol-vinyl gelation system for fabricating cell-instructive hydrogels. Gelation was achieved via a mixed-mode step-and-chain-growth photopolymerization using functionalized 4-arm poly(ethylene glycol) as backbone macromer, eosin-Y as photosensitizer, and di-thiol containing molecule as dual purpose co-initiator/cross-linker. N-vinylpyrrolidone (NVP) was used to accelerate gelation kinetics and to adju...

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

    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

  12. Influence of electrical stimulation on 3D-cultures of Adipose Tissue Derived Progenitor Cells (ATDPCs) behavior

    Castells-Sala, C.; Sánchez Terrones, Benjamín; Recha-Sancho, L.; V. Puig; Bragós Bardia, Ramon; C. E. Semino

    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 manuscr...

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

    Caterina Morabito

    2015-01-01

    Full Text Available 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.

  14. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    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.

  15. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    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. (paper)

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

    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

  17. Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

    Christian Bucher

    2013-01-01

    Full Text Available Intervertebral disc (IVD cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5 by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

  18. Sub-100 nm biodegradable nanoparticles: in vitro release features and toxicity testing in 2D and 3D cell cultures

    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. (paper)

  19. CARS and SHG microscopy to follow the collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin gel 3D cultures

    Mortati, Leonardo; Sassi, Maria Paola

    2011-01-01

    Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with second harmonic generation (SHG) technique in order to follow the early stage of stem cell differentiation within a 3D scaffold. CARS microscopy can detect lipid membranes and droplet compartments in living cells and SHG microscopy enables a strong imaging contrast for molecules with a non-centrosymmetric ordered structure like collagen. One of the first evidence of hMSCs differentiation is the formation of an extracellular matrix (ECM) where the collagen protein is its main component. This work demonstrated the multimodal CARS and SHG microscopy as a powerful non-invasive label free technique to investigate the collagen production dynamic in living cell 3D cultures. Its ability to image the cell morphology and the produced collagen distribution on a long term (4 weeks) experiment allowed to obtain important information about the cell-scaffold interaction and the ECM production. The very low limit reached in detecting collagen has permit...

  20. Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer.

    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

  1. Functional Interactions between 17β-Estradiol and Progesterone Regulate Autophagy during Acini Formation by Bovine Mammary Epithelial Cells in 3D Cultures

    Katarzyna Zielniok

    2014-01-01

    Full Text Available Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2 and progesterone (P4. Mammary epithelial cells (MECs cultured on reconstituted basement membrane (rBM form three-dimensional (3D acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR involved in autophagy induction.

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

    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 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 (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

  3. Reduced cytotoxicity and enhanced bioactivity of cationic antimicrobial peptides liposomes in cell cultures and 3D epidermis model against HSV.

    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. Tailored and biodegradable poly(2-oxazoline) microbeads as 3D matrices for stem cell culture in regenerative therapies.

    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

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

    W. Schuhr

    2015-08-01

    Niepce (1827, but seem to promise a great future also in 3D Cultural Heritage documentation. *Last not least 3D printers more and more seem to conquer the IT-market, obviously showing an international competition.

  6. Real-time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor.

    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 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:26202449

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

    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

  8. Dextran and Polymer Polyethylene Glycol (PEG Coating Reduce Both 5 and 30 nm Iron Oxide Nanoparticle Cytotoxicity in 2D and 3D Cell Culture

    Alisa Morss Clyne

    2012-05-01

    Full Text Available Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG. Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.

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

    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

  10. Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions

    Rath, Subha N.; Strobel, Leonie A; Arkudas, Andreas; Beier, Justus P.; Maier, Anne-Kathrin; Greil, Peter; Horch, Raymund E.; Kneser, Ulrich

    2012-01-01

    In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary ra...

  11. Integration of porosity and bio-functionalization to form a 3D scaffold: cell culture studies and in vitro degradation

    In this study, porous poly(lactide-co-glycolide) (PLGA) (50/50) microspheres have been fabricated by the gas-foaming technique using ammonium bicarbonate as a gas-foaming agent. Microspheres of different porosities have been formulated by varying the concentration of the gas-foaming agent (0%, 5%, 10% and 15% w/v). These microspheres were characterized for particle size, porosity and average pore size, morphology, water uptake ratio and surface area and it was found that the porosity, pore size and surface area increased on increasing the concentration of the gas-foaming agent. Further, the effect of porosity on degradation behavior was evaluated over a 12 week period by measuring changes in mass, pH, molecular weight and morphology. Porosity was found to have an inverse relationship with degradation rate. To render the surface of the microspheres biomimetic, peptide P-15 was coupled to the surface of these microspheres. In vitro cell viability, proliferation and morphological evaluation were carried out on these microsphere scaffolds using MG-63 cell line to study the effect of the porosity and pore size of scaffolds and to evaluate the effect of P-15 on cell growth on porous scaffolds. MTT assay, actin, alizarin staining and SEM revealed the potential of biomimetic porous PLGA (50/50) microspheres as scaffolds for tissue engineering. As shown in graphical representation, an attempt has been made to correlate the cell behavior on the scaffolds (growth, proliferation and cell death) with the concurrent degradation of the porous microsphere scaffold as a function of time.

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

    Debora B. Petropolis

    2014-04-01

    Full Text Available 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.

  13. Stem cell reprogramming: A 3D boost

    Abilez, Oscar J.; Wu, Joseph C.

    2016-03-01

    Biophysical factors in an optimized three-dimensional microenvironment enhance the reprogramming efficiency of human somatic cells into pluripotent stem cells when compared to traditional cell-culture substrates.

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

    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

  15. Secondary activation of c-abl may be related to translocation to the nucleolar organizer region in an in vitro cultured rat leukemia cell line (K3D)

    Localization of cellular oncogenes (c-onc) near the break points of translocations in tumor cells has indicated involvement of these genes in neoplastic growth. Enhanced transcription of the cellular homolog (c-abl) of the transforming sequence of Abelson murine leukemia virus was observed in K3D, which was one of the cloned cell lines of 7,12-dimethylbenz[a]anthracene-induced rat erythroblastic leukemia. Since the c-abl activation was not observed in the parent cell line (K2D) from which K3D was derived and the latter was different from the former in the presence of a new marker chromosome, t(3;12), this marker may play a role in the expression of c-abl in K3D cells. In contrast to the human c-onc assignments, few rat c-onc assignments have been reported. In situ molecular hybridization studies assigned c-abl to the 3q12 site of the normal chromosome 3 and to the break point of the translocation t(3;12) in K3D cells. Another break point in this translocation chromosome 12p11 involves the nucleolar region, and the 3;12 translocation may involve c-abl and nucleolar cistrons. A cloned v-abl-specific probe was used for hybridization and labelled with 125I-dCTP. These results provide evidence of secondary c-onc activation during karyotypic evolution of cloned malignant cells

  16. EXPLORING CULTURAL HERITAGE RESOURCES IN A 3D COLLABORATIVE ENVIRONMENT

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

    2012-01-01

    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 ex...

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

    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.

  18. Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

    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

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

    Simon, Karen Alambra; Warren, Nicholas J.; Mosadegh, Bobak; Mohammady, Marym R.; Whitesides, George McClelland; Armes, Steven P.

    2015-01-01

    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,...

  20. Reprogramming mediated radio-resistance of 3D-grown cancer cells

    In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. (author)

  1. Development, Characterization and Cell Cultural Response of 3D Biocompatible Micro-Patterned Poly-ε-Caprolactone Scaffolds Designed and Fabricated Integrating Lithography and Micromolding Fabrication Techniques

    Limongi, Tania

    2014-12-12

    Scaffold design and fabrication are very important subjects for biomaterial, tissue engineering and regenerative medicine research playing a unique role in tissue regeneration and repair. Among synthetic biomaterials Poly-ε- Caprolactone (PCL) is very attractive bioresorbable polyester due to its high permeability, biodegradability and capacity to be blended with other biopolymers. Thanks to its ability to naturally degrade in tissues, PCL has a great potential as a new material for implantable biomedical micro devices. This work focuses on the establishment of a micro fabrication process, by integrating lithography and micromolding fabrication techniques, for the realization of 3D microstructure PCL devices. Scaffold surface exhibits a combination in the patterned length scale; cylindrical pillars of 10 μm height and 10 μm diameter are arranged in a hexagonal lattice with periodicity of 30 μm and their sidewalls are nano-sculptured, with a regular pattern of grooves leading to a spatial modulation in the z direction. In order to demonstrate that these biocompatible pillared PCL substrates are suitable for a proper cell growth, NIH/3T3 mouse embryonic fibroblasts were seeded on them and cells key adhesion parameters were evaluated. Scanning Electron Microscopy and immunofluorescence analysis were carried out to check cell survival, proliferation and adhesion; cells growing on the PCL substrates appeared healthy and formed a well-developed network in close contact with the micro and nano features of the pillared surface. Those 3D scaffolds could be a promising solution for a wide range of applications within tissue engineering and regenerative medicine applications.

  2. Galpha/LGN-mediated asymmetric spindle positioning does not lead to unequal cleavage of the mother cell in 3-D cultured MDCK cells

    Xiao, Zhuoni; Wan, Qingwen; Du, Quansheng; Zheng, Zhen

    2012-01-01

    The position of the mitotic spindle plays a key role in spatial control of cell division. It is generally believed that when a spindle is positioned asymmetrically in a dividing cell, the resulting daughter cells are usually unequal in size due to eccentric cleavage of the mother cell. Molecular mechanisms underlying the generation of unequal sized daughter cells have been extensively studied in Drosophila neuroblast and C elegans zygote where the Gα subunit of the heterotrimeric G proteins a...

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

    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

  4. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-01-01

    Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering. PMID:27321481

  5. Radiobiology goes 3D: How ECM and cell morphology impact on cell survival after irradiation

    Translational research is essential to find new therapeutic approaches to improve cancer patient survival. Despite extensive efforts in preclinical studies, many novel therapies fail to turn out to be translational from bench to beside. Therefore, new models better reflecting the conditions in vivo are needed to generate results, which transfer reliably into the clinic. The use of three-dimensional (3D) cell culture models has provided new emerging insights into the understanding of cellular behavior upon cancer therapies. Interestingly, cells cultured in a 3D extracellular matrix are more radio- and chemoresistant than cells grown under conventional 2D conditions. In this review, we summarize and discuss underlying mechanisms of this phenomenon including integrin-mediated cell-matrix interactions, cell shape, nuclear organization and chromatin structure. Identifying the molecular differences between 2D and 3D cultured cells will offer the opportunity to improve our research and widen our therapeutic possibilities against cancer.

  6. Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D) human cell culture model of oral mucositis

    Lambros, Maria P.; DeSalvo, Michael K.; Jonathan Moreno; Hari Chandana Mulamalla; Lavanya Kondapalli

    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 cystein...

  7. Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

    Hasselbach, Laura A.; Susan M. Irtenkauf; Lemke, Nancy W.; Nelson, Kevin K.; Artem D. Berezovsky; Carlton, Enoch T.; Andrea D. Transou; Mikkelsen, Tom; deCarvalho, Ana C.

    2014-01-01

    Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated i...

  8. 3D visualization and virtual reality for cultural heritage diagnostic

    Lucio Colizzi

    2007-07-01

    Full Text Available In the past years, many different new technologies for Cultural Heritage Diagnostic have been developed. In particular laser scanner surveys with digital photogrammetry and also multi-spectral surveys are becoming very useful and inalienable tools for non invasive diagnosis. In the SIDART Project (Integrated System for Cultural Heritage diagnosis, we develop a software able to visualize and elaborate triangulated surfaces coming from high resolution laser scanner survey. In this paper, we want to present the most innovative aspect of our study, that is the possibility to visualize and work in default mode or in immersive Stereoscopy (3D mode. This lets the operator perceive the third dimension and the “virtual investigation” of the object becomes more realistic. This lets us take into consideration in a more simple, natural and correct way and also reduce the possibility to make wrong evaluation due to the false prospective of the classic visualization.

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

    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. DIACHRONIC 3D RECONSTRUCTION FOR LOST CULTURAL HERITAGE

    G. Guidi

    2012-09-01

    Full Text Available 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.

  11. Diachronic 3d Reconstruction for Lost Cultural Heritage

    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. 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.

    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

  13. Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D) human cell culture model of oral mucositis.

    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

  14. Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D human cell culture model of oral mucositis

    Maria P. Lambros

    2015-12-01

    Full Text Available 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.

  15. 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation.

    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

  16. Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

    Yinzhi Lai

    Full Text Available Cells cultured in three dimensional (3D scaffolds as opposed to traditional two-dimensional (2D substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate. Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.

  17. Screening for Stromal and Matrix Effects in 3D Microenvironments of Breast Cancer Cells

    Montanez-Sauri, Sara I.

    Breast cancer progression ensures through the acquisition of genetic mutations, the uncontrollable growth of cells, and their progression to invasion. Studies have shown that the surrounding three-dimensional (3D) microenvironment can also influence breast cancer cell progression by controlling the morphology, differentiation, proliferation, and migration of cells. However, most of the currently available in vitro screening platforms are based on the two-dimensional (2D) culture of cells, and do not provide cells with the complex 3D microenvironment that exists in vivo. Therefore, there is a need for more biologically relevant in vitro platforms to help decipher the complexity of the microenvironment and its influence in breast cancer. In this dissertation we present an automated microfluidic platform that allows to efficiently screen for the effect of multiple matrix and stromal microenvironment in 3D cultures of breast cancer cells. Several extracellular matrix (ECM) compositions and stromal cells are included in the 3D microenvironments to examine their influence on breast cancer cell behavior. The screening results suggest that collagen gels with fibronectin might be influencing paracrine signals between breast cancer cells and stromal cells. The ability of the platform to culture and treat cells in 3D microenvironments offers a powerful screening tool for the identification of compounds and interactions using more in vivo-like 3D microenvironments. The identification of these mechanisms will increase our current understanding of breast cancer, and will aid in the identification of potential therapeutics.

  18. Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

    Fey, S. J.; Wrzesinski, Krzysztof

    2012-01-01

    Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this...

  19. Laser printing of cells into 3D scaffolds

    One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.

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

    W. Schuhr; Lee, J. D.

    2015-01-01

    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 existin...

  1. Cyto-3D-print to attach mitotic cells.

    Castroagudin, Michelle R; Zhai, Yujia; Li, Zhi; Marnell, Michael G; Glavy, Joseph S

    2016-08-01

    The Cyto-3D-print is an adapter that adds cytospin capability to a standard centrifuge. Like standard cytospinning, Cyto-3D-print increases the surface attachment of mitotic cells while giving a higher degree of adaptability to other slide chambers than available commercial devices. The use of Cyto-3D-print is cost effective, safe, and applicable to many slide designs. It is durable enough for repeated use and made of biodegradable materials for environment-friendly disposal. PMID:26464272

  2. Characteristics of tumor and host cells in 3-D simulated microgravity environment

    Chopra, V.; Dinh, T.; Wood, T.; Pellis, N.; Hannigan, E.

    Co-cultures of three-dimensional (3-D) constructs of one cell type with dispersed cells of a second cell type in low-shear rotating suspension cultures in simulated microgravity environment have been used to investigate invasive properties of normal and malignant cell types. We have shown that the epithelial and endothelial cells undergo a switch in characteristics when grown in an in vitro 3-D environment, that mimics the in vivo host environment as compared with conventional two-dimensional (2-D) monolayer cultures. Histological preparations and immunohistochemical staining procedures of cocultured harvests demonstrated various markers of interest: like collagen vimentin, mucin, elastin, fibrin, fibrinogen, cytokeratin, adhesion molecules and various angiogenic factors by tumor cells from gynecological cancer patients along with fibroblasts, endothelial cells and patient-derived mononuclear cells (n=8). The growth rate was enhanced 10-15 folds by 3-D cocultures of patient-derived cells as compared with 2-D monolayer cultures and 3-D monocultures. The production of interleukin-2, interleukin-6, interleukin -8, vascular endothelial cell growth factor, basic fibroblast growth factor, and angiogenin was studied by using ELISA and RT- PCR. Human umbilical vein-derived endothelial cell (HUVEC) were used to study the mitogenic response of the conditioned medium collected from 3-D monocultures and cocultures during proliferation and migration assays. The conditioned medium collected from 3-D cocultures of cancer cells also 1) increased the expression of message levels of vascular endothelial growth factor and its receptor flt-1 and KDR was observed by HUVEC, and 2) increased the expression of intracellular and vascular cell adhesion molecules on the surface of HUVEC, when measured by using Live cell ELISA assays and immunofluorescent staining as compared with 3-D monocultures of normal epithelial cells. There was an increase in production of 1) enzymatic activity that

  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.

    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. Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

    Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

    2014-02-01

    Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

  5. Fibroblasts Lead the Way: A Unified View of 3D Cell Motility.

    Petrie, Ryan J; Yamada, Kenneth M

    2015-11-01

    Primary human fibroblasts are remarkably adaptable, able to migrate in differing types of physiological 3D tissue and on rigid 2D tissue culture surfaces. The crawling behavior of these and other vertebrate cells has been studied intensively, which has helped generate the concept of the cell motility cycle as a comprehensive model of 2D cell migration. However, this model fails to explain how cells force their large nuclei through the confines of a 3D matrix environment and why primary fibroblasts can use more than one mechanism to move in 3D. Recent work shows that the intracellular localization of myosin II activity is governed by cell-matrix interactions to both force the nucleus through the extracellular matrix (ECM) and dictate the type of protrusions used to migrate in 3D. PMID:26437597

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

    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

  7. Cell force measurements in 3D microfabricated environments based on compliant cantilevers.

    Marelli, Mattia; Gadhari, Neha; Boero, Giovanni; Chiquet, Matthias; Brugger, Jürgen

    2014-01-21

    We report the fabrication, functionalization and testing of microdevices for cell culture and cell traction force measurements in three-dimensions (3D). The devices are composed of bent cantilevers patterned with cell-adhesive spots not lying on the same plane, and thus suspending cells in 3D. The cantilevers are soft enough to undergo micrometric deflections when cells pull on them, allowing cell forces to be measured by means of optical microscopy. Since individual cantilevers are mechanically independent of each other, cell traction forces are determined directly from cantilever deflections. This proves the potential of these new devices as a tool for the quantification of cell mechanics in a system with well-defined 3D geometry and mechanical properties. PMID:24217771

  8. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

    Highlights: ► Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. ► Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. ► 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 μm porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

  9. 3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells.

    Tong Luo

    Full Text Available The 3D geometry of individual vascular smooth muscle cells (VSMCs, which are essential for understanding the mechanical function of blood vessels, are currently not available. This paper introduces a new 3D segmentation algorithm to determine VSMC morphology and orientation.A total of 112 VSMCs from six porcine coronary arteries were used in the analysis. A 3D semi-automatic segmentation method was developed to reconstruct individual VSMCs from cell clumps as well as to extract the 3D geometry of VSMCs. A new edge blocking model was introduced to recognize cell boundary while an edge growing was developed for optimal interpolation and edge verification. The proposed methods were designed based on Region of Interest (ROI selected by user and interactive responses of limited key edges. Enhanced cell boundary features were used to construct the cell's initial boundary for further edge growing. A unified framework of morphological parameters (dimensions and orientations was proposed for the 3D volume data. Virtual phantom was designed to validate the tilt angle measurements, while other parameters extracted from 3D segmentations were compared with manual measurements to assess the accuracy of the algorithm. The length, width and thickness of VSMCs were 62.9±14.9 μm, 4.6±0.6 μm and 6.2±1.8 μm (mean±SD. In longitudinal-circumferential plane of blood vessel, VSMCs align off the circumferential direction with two mean angles of -19.4±9.3° and 10.9±4.7°, while an out-of-plane angle (i.e., radial tilt angle was found to be 8±7.6° with median as 5.7°.A 3D segmentation algorithm was developed to reconstruct individual VSMCs of blood vessel walls based on optical image stacks. The results were validated by a virtual phantom and manual measurement. The obtained 3D geometries can be utilized in mathematical models and leads a better understanding of vascular mechanical properties and function.

  10. XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

    Taylor-Kashton, Cheryl; Lichtensztejn, Daniel; Baloglu, Erkan; Senapedis, William; Shacham, Sharon; Kauffman, Michael G; Kotb, Rami; Mai, Sabine

    2016-12-01

    Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991404

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

    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

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

    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.

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

    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

  14. 3D culture of ovarian follicles: a system towards their engineering?

    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

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

    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

  16. Development and Validation of a 3D Clinostat for the Study of Cells during Microgravity Simulation.

    Russomano, T; Cardoso, R; Falcao, F; Dalmarco, G; V Dos Santos, C; F Dos Santos, L; G de Azevedo, D; Dos Santos, M; Martinelli, L; Motta, J; Forraz, N; McGuckin, C

    2005-01-01

    The clinostat was originally used to find out why plant roots appear to grow predominantly toward the center of the Earth. Over the last 2-3 decades, slow- and fast-rotating 2D and 3D clinostats have been used to assess cellular adaptation to this environment. A cell culture is placed in a spin module of the clinostat platform and its rotation is set empirically (2-3 rpm). The machine is then allowed to run for a specified period (hours to days) after which the cultures are removed and assayed for specific properties, such as cell growth, size and shape, distribution of receptors, integrity of the cytoskeleton or gene expression. A 3D clinostat was developed by the Microgravity Laboratory/IPCT-PUCRS group and validated by the Stem Cell Group of Kingston University London, which used 4 different types of human cancer cells and cord blood stem cells (CBSC). After rotation for 19h at 37degC, 5%CO2 humidified atmosphere, the 3D clinostat significantly improved proliferation potential of all tested cell populations when compared to static cultures. After only 5 days, high definition microscopic analysis revealed that all CBSC adhered and expanded onto the BDtrade 3D collagen composite scaffolds, and cross-developed into hepatocyte-like cells upon stimulation. PMID:17282243

  17. Alteration of cellular behavior and response to PI3K pathway inhibition by culture in 3D collagen gels.

    Brian Fallica

    Full Text Available Most investigations into cancer cell drug response are performed with cells cultured on flat (2D tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D extracellular matrix (ECM is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.

  18. Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

    Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

    2016-08-01

    Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. PMID:27252227

  19. 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

    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 ...

  20. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation

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

    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.

  2. A novel mechanotactic 3D modeling of cell morphology

    Cell morphology plays a critical role in many biological processes, such as cell migration, tissue development, wound healing and tumor growth. Recent investigations demonstrate that, among other stimuli, cells adapt their shapes according to their substrate stiffness. Until now, the development of this process has not been clear. Therefore, in this work, a new three-dimensional (3D) computational model for cell morphology has been developed. This model is based on a previous cell migration model presented by the same authors. The new model considers that during cell–substrate interaction, cell shape is governed by internal cell deformation, which leads to an accurate prediction of the cell shape according to the mechanical characteristic of its surrounding micro-environment. To study this phenomenon, the model has been applied to different numerical cases. The obtained results, which are qualitatively consistent with well-known related experimental works, indicate that cell morphology not only depends on substrate stiffness but also on the substrate boundary conditions. A cell located within an unconstrained soft substrate (several kPa) with uniform stiffness is unable to adhere to its substrate or to send out pseudopodia. When the substrate stiffness increases to tens of kPa (intermediate and rigid substrates), the cell can adequately adhere to its substrate. Subsequently, as the traction forces exerted by the cell increase, the cell elongates and its shape changes. Within very stiff (hard) substrates, the cell cannot penetrate into its substrate or send out pseudopodia. On the other hand, a cell is found to be more elongated within substrates with a constrained surface. However, this elongation decreases when the cell approaches it. It can be concluded that the higher the net traction force, the greater the cell elongation, the larger the cell membrane area, and the less random the cell alignment. (paper)

  3. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. PMID:23085529

  4. The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments.

    Krzysztof Wrzesinski

    Full Text Available 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.

  5. 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: activating enzymes (Phase I).

    Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Blatz, Veronika; Jäckh, Christine; Freytag, Eva-Maria; Fabian, Eric; Landsiedel, Robert; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

    2012-05-01

    Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDerm™ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing. PMID:22509833

  6. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Z. Wen

    2013-08-01

    Full Text Available Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  7. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer

  8. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Wen, Z.; Liao, Q.; Hu, Y.; You, L.; Zhou, L.; Zhao, Y. [Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, Beijing (China)

    2013-08-10

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  9. 3D MODELLING AND INTERACTIVE WEB-BASED VISUALIZATION OF CULTURAL HERITAGE OBJECTS

    Koeva, M. N.

    2016-01-01

    Nowadays, there are rapid developments in the fields of photogrammetry, laser scanning, computer vision and robotics, together aiming to provide highly accurate 3D data that is useful for various applications. In recent years, various LiDAR and image-based techniques have been investigated for 3D modelling because of their opportunities for fast and accurate model generation. For cultural heritage preservation and the representation of objects that are important for tourism and their interact...

  10. 3D Modeling from Multi-views Images for Cultural Heritage in Wat-Pho, Thailand

    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.

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

    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

  12. Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

    Gartia, Manas Ranjan [Department of Nuclear, Plasma and Radiological Engineering, University of Illinois, Urbana, IL 61801 (United States); Hsiao, Austin; Logan Liu, G [Department of Bioengineering, University of Illinois, Urbana, IL 61801 (United States); Sivaguru, Mayandi [Institute for Genomic Biology, University of Illinois, Urbana, IL 61801 (United States); Chen Yi, E-mail: loganliu@illinois.edu [Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801 (United States)

    2011-09-07

    We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.

  13. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    Lee, Wonjae; Park, Jon

    2016-07-01

    Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues.

  14. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    Lee, Wonjae; Park, Jon

    2016-01-01

    Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues. PMID:27381562

  15. Metadata and Tools for Integration and Preservation of Cultural Heritage 3D Information

    Achille Felicetti

    2011-12-01

    Full Text Available In this paper we investigate many of the various storage, portability and interoperability issues arising among archaeologists and cultural heritage people when dealing with 3D technologies. On the one side, the available digital repositories look often unable to guarantee affordable features in the management of 3D models and their metadata; on the other side the nature of most of the available data format for 3D encoding seem to be not satisfactory for the necessary portability required nowadays by 3D information across different systems. We propose a set of possible solutions to show how integration can be achieved through the use of well known and wide accepted standards for data encoding and data storage. Using a set of 3D models acquired during various archaeological campaigns and a number of open source tools, we have implemented a straightforward encoding process to generate meaningful semantic data and metadata. We will also present the interoperability process carried out to integrate the encoded 3D models and the geographic features produced by the archaeologists. Finally we will report the preliminary (rather encouraging development of a semantic enabled and persistent digital repository, where 3D models (but also any kind of digital data and metadata can easily be stored, retrieved and shared with the content of other digital archives.

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

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

    2016-01-01

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

  17. 三维环境下肌源性干细胞分化为胰岛样细胞团%Muscle-derived stem cells differentiate into the islet cells in 3D culture

    殷甜甜; 刘畅; 梅晰凡; 王滢丽

    2013-01-01

    Objective: To study the effect of the extracellular microenvironment in muscle-derived stem cells (MDSCs) differentiating into the islet cells. Methods: MDSCs were extracted by mixed enzymatic digestion, purified by differential adherent culture, and induced by the cells after split into following groups: a collagen and chemical group, a chemical group and a control group. The differentiation was induced separately and the cell morphology was observed simultaneously. Insulin producing cell clusters (IPCCs) were identified by dithizone (DTZ) staining, immunocytochemical staining were used to examine the production of insulin glucagons etc and reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression.Results: Under an ordinary light microscope, the size of the islet cells induced by the collagen and chemical group was bigger than that in the chemical group; and the DTZ dyeing results showed that the induced islet cells of the collagen and chemical group secreted more insulin than did those of the chemical group; immunocytochemical staining showed that the induced islet cells in the collagen and chemical group were more similar to adult rat islet tissues, and detected by RT-polymerase chain reaction, the gene expression of the induced islet cells in the collagen and chemical group was higher than that in the chemical group. Conclusion: In the three dimensional environment, muscle stem cells could differentiate into the pancreatic islet cells, and muscle source cells can be differentiated into bigger and more mature islet cells.%目的:运用三维细胞培养模型研究细胞外微环境对肌源性干细胞分化为胰岛样细胞的作用.方法:用差速贴壁的方法对SD大鼠肌源性干细胞进行分离和培养,将细胞分为胶原支架化学诱导剂组,化学诱导剂组,空白组.分别观察诱导后细胞的生长形态,采用双巯腙DTZ染色鉴定胰岛素分泌细胞,细胞免疫化学检测胰岛相关蛋

  18. Results of comparative RBMK neutron computation using VNIIEF codes (cell computation, 3D statics, 3D kinetics). Final report

    In conformity with the protocol of the Workshop under Contract open-quotes Assessment of RBMK reactor safety using modern Western Codesclose quotes VNIIEF performed a neutronics computation series to compare western and VNIIEF codes and assess whether VNIIEF codes are suitable for RBMK type reactor safety assessment computation. The work was carried out in close collaboration with M.I. Rozhdestvensky and L.M. Podlazov, NIKIET employees. The effort involved: (1) cell computations with the WIMS, EKRAN codes (improved modification of the LOMA code) and the S-90 code (VNIIEF Monte Carlo). Cell, polycell, burnup computation; (2) 3D computation of static states with the KORAT-3D and NEU codes and comparison with results of computation with the NESTLE code (USA). The computations were performed in the geometry and using the neutron constants presented by the American party; (3) 3D computation of neutron kinetics with the KORAT-3D and NEU codes. These computations were performed in two formulations, both being developed in collaboration with NIKIET. Formulation of the first problem maximally possibly agrees with one of NESTLE problems and imitates gas bubble travel through a core. The second problem is a model of the RBMK as a whole with imitation of control and protection system controls (CPS) movement in a core

  19. State-of-the-Art of 3D Cultures (Organs-on-a-Chip) in Safety Testing and Pathophysiology

    Alepee, Natalie; Bahinski, Anthony; Daneshian, Mardas; De Weyer, Bart; Fritsche, Ellen; Goldberg, Alan; Hansmann, Jan; Hartung, Thomas; Haycock, John; Hogberg, Helena T.; Hoelting, Lisa; Jens M Kelm; Kadereit, Suzanne; McVey, Emily; Landsiedel, Robert

    2015-01-01

    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. An important building block of this strategy that has emerged during the last years are threedimensional (3D) cell culture models. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ ...

  20. File list: Pol.ALL.20.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.20.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX301459,SRX373...73041 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.20.Polr3d.AllCell.bed ...

  1. File list: Pol.ALL.05.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.05.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX373040,SRX373...04148 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.05.Polr3d.AllCell.bed ...

  2. File list: Pol.ALL.50.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.50.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX301459,SRX373...04147 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.50.Polr3d.AllCell.bed ...

  3. File list: Pol.ALL.10.Polr3d.AllCell [Chip-atlas[Archive

    Full Text Available Pol.ALL.10.Polr3d.AllCell mm9 RNA polymerase Polr3d All cell types SRX373040,SRX301...04147 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.10.Polr3d.AllCell.bed ...

  4. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

  5. Oscillating Cell Culture Bioreactor

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  6. Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells.

    Karl Gledhill

    Full Text Available The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.

  7. Heritable Genetic Changes in Cells Recovered From Irradiated 3D Tissue Constructs

    Michael Cornforth

    2012-03-26

    Combining contemporary cytogenetic methods with DNA CGH microarray technology and chromosome flow-sorting increases substantially the ability to resolve exchange breakpoints associated with interstitial deletions and translocations, allowing the consequences of radiation damage to be directly measured at low doses, while also providing valuable insights into molecular mechanisms of misrepair processes that, in turn, identify appropriate biophysical models of risk at low doses. Specific aims apply to cells recovered from 3D tissue constructs of human skin and, for the purpose of comparison, the same cells irradiated in traditional 2D cultures. The project includes research complementary to NASA/HRP space radiation project.

  8. Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

    Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula

    2016-08-01

    Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application. PMID:25609254

  9. Engineering a perfusable 3D human liver platform from iPS cells.

    Schepers, Arnout; Li, Cheri; Chhabra, Arnav; Seney, Benjamin Tschudy; Bhatia, Sangeeta

    2016-07-01

    In vitro models of human tissue are crucial to our ability to study human disease as well as develop safe and effective drug therapies. Models of single organs in static and microfluidic culture have been established and shown utility for modeling some aspects of health and disease; however, these systems lack multi-organ interactions that are critical to some aspects of drug metabolism and toxicity. Thus, as part of a consortium of researchers, we have developed a liver chip that meets the following criteria: (1) employs human iPS cells from a patient of interest, (2) cultures cells in perfusable 3D organoids, and (3) is robust to variations in perfusion rate so as to be compatible in series with other specialized tissue chips (e.g. heart, lung). In order to achieve this, we describe methods to form hepatocyte aggregates from primary and iPS-derived cells, alone and in co-culture with support cells. This necessitated a novel culture protocol for the interrupted differentiation of iPS cells that permits their removal from a plated surface and aggregation while maintaining phenotypic hepatic functions. In order to incorporate these 3D aggregates in a perfusable platform, we next encapsulated the cells in a PEG hydrogel to prevent aggregation and overgrowth once on chip. We adapted a C-trap chip architecture from the literature that enabled robust loading with encapsulated organoids and culture over a range of flow rates. Finally, we characterize the liver functions of this iHep organoid chip under perfusion and demonstrate a lifetime of at least 28 days. We envision that such this strategy can be generalized to other microfluidic tissue models and provides an opportunity to query patient-specific liver responses in vitro. PMID:27296616

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

    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

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

    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

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

    Kim, Sun-Ah, E-mail: j.sarah.k@gmail.com [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Kuh, Hyo-Jeong, E-mail: hkuh@catholic.ac.kr [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of)

    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.

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

    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.

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

    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

  15. 3D-Printing Crystallographic Unit Cells for Learning Materials Science and Engineering

    Rodenbough, Philip P.; Vanti, William B.; Chan, Siu-Wai

    2015-01-01

    Introductory materials science and engineering courses universally include the study of crystal structure and unit cells, which are by their nature highly visual 3D concepts. Traditionally, such topics are explored with 2D drawings or perhaps a limited set of difficult-to-construct 3D models. The rise of 3D printing, coupled with the wealth of…

  16. Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha; Gehl, Julie; Rols, Marie-Pierre

    2015-01-01

    Background Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy–and normal cell sensitivity. Methods Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different h...

  17. The potential of 3D techniques for cultural heritage object documentation

    Bitelli, Gabriele; Girelli, Valentina A.; Remondino, Fabio; Vittuari, Luca

    2007-01-01

    The generation of 3D models of objects has become an important research point in many fields of application like industrial inspection, robotics, navigation and body scanning. Recently the techniques for generating photo-textured 3D digital models have interested also the field of Cultural Heritage, due to their capability to combine high precision metrical information with a qualitative and photographic description of the objects. In fact this kind of product is a fundamental support for documentation, studying and restoration of works of art, until a production of replicas by fast prototyping techniques. Close-range photogrammetric techniques are nowadays more and more frequently used for the generation of precise 3D models. With the advent of automated procedures and fully digital products in the 1990s, it has become easier to use and cheaper, and nowadays a wide range of commercial software is available to calibrate, orient and reconstruct objects from images. This paper presents the complete process for the derivation of a photorealistic 3D model of an important basalt stela (about 70 x 60 x 25 cm) discovered in the archaeological site of Tilmen Höyük, in Turkey, dating back to 2nd mill. BC. We will report the modeling performed using passive and active sensors and the comparison of the achieved results.

  18. Induced tauopathy in a novel 3D-culture model mediates neurodegenerative processes: a real-time study on biochips.

    Diana Seidel

    Full Text Available Tauopathies including Alzheimer's disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer's disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner.Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer's disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy.Bringing together our novel complex 3

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

    G Bouet

    2015-05-01

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

  20. Effect of biomimetic 3D environment of an injectable polymeric scaffold on MG-63 osteoblastic-cell response

    Solid PLGA microspheres were fabricated and characterized in terms of their in vitro degradation behaviour. Microsphere scaffolds were then modified covalently by P-15 (GTPGPQGIAGQRGVV) to obtain a 3D bioactive collagen surrogate matrix for bone filling applications. These scaffolds were characterized for surface topography, hydrophilicity and evaluated for their effect on osteoblastic activity of MG-63 cell line vis-a-vis 2D monolayer culture. AFM and contact angle experiments indicated enhanced nano-level roughness and hydrophilicity on P-15 modification. Modified scaffolds showed enhanced cell attachment, proliferation, extracellular matrix formation, mineralization and collagen type-I expression when compared to unmodified microspheres, prerequisite for bone filling applications. On long term in vitro cell culture, however, decreased cell viability was observed which may be attributed to the acidic microenvironment generated due to polymer degradation and reduction in nutrient diffusion through the copious ECM formed in 3D scaffolds. Though a higher cell count could be obtained in 2D monolayer cell culture, it was overshadowed by weak cell attachment, poor phenotypic characteristics, decreased cell viability and low mineralization levels, over 28 day cell culture studies. Results indicate that P-15 modified microsphere scaffolds may provide a natural, biomimetic 3D environment and may be successfully exploited for non-invasive bone filling applications.

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

    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

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

    Foroni, Laura [Pathology Unit, Department of Haematology, Oncology and Clinical Pathology, S. Orsola-Malpighi Hospital, Bologna University (Italy); Vasuri, Francesco, E-mail: vasurifrancesco@libero.it [Pathology Unit, Department of Haematology, Oncology and Clinical Pathology, S. Orsola-Malpighi Hospital, Bologna University (Italy); Chair of Vascular Surgery, Department of Specialistic Surgery and Anaesthesiological Sciences, S. Orsola-Malpighi Hospital, Bologna University (Italy); Valente, Sabrina [Pathology Unit, Department of Haematology, Oncology and Clinical Pathology, S. Orsola-Malpighi Hospital, Bologna University (Italy); Gualandi, Chiara [Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, RU Bologna), Bologna University (Italy); Focarete, Maria Letizia [Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, RU Bologna), Bologna University (Italy); Health Science and Technologies–Interdepartmental Center for Industrial Research (HST-ICIR), Bologna University (Italy); Caprara, Giacomo [Pathology Unit, Department of Haematology, Oncology and Clinical Pathology, S. Orsola-Malpighi Hospital, Bologna University (Italy); Scandola, Mariastella [Department of Chemistry “G. Ciamician” and National Consortium of Materials Science and Technology (INSTM, RU Bologna), Bologna University (Italy); D' Errico-Grigioni, Antonia; Pasquinelli, Gianandrea [Pathology Unit, Department of Haematology, Oncology and Clinical Pathology, S. Orsola-Malpighi Hospital, Bologna University (Italy)

    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.

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

    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

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

    Bashar Alsadik

    2014-03-01

    Full Text Available 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.

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

    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

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

    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

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

    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 (105-108 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% (CT50) using commercially available drugs which further correlated well with published in vivo LD50 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 multiple cell types.

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

    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),...

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

    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

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

    Anna Acheva

    Full Text Available 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.

  11. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

  12. Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system.

    Seo, Ha-Rim; Jeong, Hyo Eun; Joo, Hyung Joon; Choi, Seung-Cheol; Park, Chi-Yeon; Kim, Jong-Ho; Choi, Ji-Hyun; Cui, Long-Hui; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2016-01-01

    The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis. PMID:27357248

  13. Microfluidics 3D gel-island chip for single cell isolation and lineage-dependent drug responses study.

    Zhang, Zhixiong; Chen, Yu-Chih; Cheng, Yu-Heng; Luan, Yi; Yoon, Euisik

    2016-07-01

    3D cell culture in the extracellular matrix (ECM), which not only provides structural support to cellular constituents, but also initiates regulatory biochemical cues for a variety of important cell functions in tissue, has become more and more important in understanding cancer pathology and drug testing. Although the ECM-gel has been used in cell culture both in bulk and on-chip, previous studies focused on collective cell behavior rather than single-cell heterogeneity. To track the behavior of each individual cell, we have developed a gel-island chip, which can form thousands of islands containing single cells encapsulated by the desired ECM. Optimized by Poisson's distribution, the device can attain 34% single cell capture efficiency of the exact number of single cells per island. A good culture media exchange rate and high cell viability can be achieved in the gel-islands. The cells in the islands can be automatically counted for high-throughput analysis. As a proof of concept, we monitored the proliferation and differentiation of single Notch+ (stem-like) T47D breast cancer cells. The 3D collagen gel environment was found to be favorable for the stem-like phenotype through better self-renewal and de-differentiation (Notch- to Notch+ transition). More interestingly, we found that the Notch- de-differentiated cells were more resistant to doxorubicin and cisplatin than the Notch+ cells. Combining the 3D ECM culture and single cell resolution, the presented platform can automatically analyze the individual cell behaviors of hundreds of cells using a small amount of drug and reagents. PMID:27270563

  14. Analyzing Biological Performance of 3D-Printed, Cell-Impregnated Hybrid Constructs for Cartilage Tissue Engineering.

    Izadifar, Zohreh; Chang, Tuanjie; Kulyk, William; Chen, Xiongbiao; Eames, B Frank

    2016-03-01

    Three-dimensional (3D) bioprinting of hybrid constructs is a promising biofabrication method for cartilage tissue engineering because a synthetic polymer framework and cell-impregnated hydrogel provide structural and biological features of cartilage, respectively. During bioprinting, impregnated cells may be subjected to high temperatures (caused by the adjacent melted polymer) and process-induced mechanical forces, potentially compromising cell function. This study addresses these biofabrication issues, evaluating the heat distribution of printed polycaprolactone (PCL) strands and the rheological property and structural stability of alginate hydrogels at various temperatures and concentrations. The biocompatibility of parameters from these studies was tested by culturing 3D hybrid constructs bioprinted with primary cells from embryonic chick cartilage. During initial two-dimensional culture expansion of these primary cells, two morphologically and molecularly distinct cell populations ("rounded" and "fibroblastic") were isolated. The biological performance of each population was evaluated in 3D hybrid constructs separately. The cell viability, proliferation, and cartilage differentiation were observed at high levels in hybrid constructs of both cell populations, confirming the validity of these 3D bioprinting parameters for effective cartilage tissue engineering. Statistically significant performance variations were observed, however, between the rounded and fibroblastic cell populations. Molecular and morphological data support the notion that such performance differences may be attributed to the relative differentiation state of rounded versus fibroblastic cells (i.e., differentiated chondrocytes vs. chondroprogenitors, respectively), which is a relevant issue for cell-based tissue engineering strategies. Taken together, our study demonstrates that bioprinting 3D hybrid constructs of PCL and cell-impregnated alginate hydrogel is a promising approach for

  15. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Sunita Nayak

    Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

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

    Wrzesinski, Krzysztof; Rogowska-Wrzesinska, Adelina; Kanlaya, Rattiyaporn;

    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...... 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...

  17. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Ansari, Nariman [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Esner, Milan; Bickle, Marc [Max Planck Institute of Molecular Cell Biology and Genetics, High-Throughput Technology Development Studio (TDS), Dresden (Germany); Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H. [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Parczyk, Karsten; Prechtl, Stefan [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Steigemann, Patrick, E-mail: Patrick.Steigemann@bayer.com [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany)

    2014-04-15

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

  18. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

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

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

    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.

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

    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.

  1. Digital microfabrication of user-defined 3D microstructures in cell-laden hydrogels.

    Soman, Pranav; Chung, Peter H; Zhang, A Ping; Chen, Shaochen

    2013-11-01

    Complex 3D interfacial arrangements of cells are found in several in vivo biosystems such as blood vasculature, renal glomeruli, and intestinal villi. Current tissue engineering techniques fail to develop suitable 3D microenvironments to evaluate the concurrent effects of complex topography and cell encapsulation. There is a need to develop new fabrication approaches that control cell density and distribution within complex 3D features. In this work, we present a dynamic projection printing process that allows rapid construction of complex 3D structures using custom-defined computer-aided-design (CAD) files. Gelatin-methacrylate (GelMA) constructs featuring user-defined spiral, pyramid, flower, and dome micro-geometries were fabricated with and without encapsulated cells. Encapsulated cells demonstrate good cell viability across all geometries both on the scaffold surface and internal to the structures. Cells respond to geometric cues individually as well as collectively throughout the larger-scale patterns. Time-lapse observations also reveal the dynamic nature of mechanical interactions between cells and micro-geometry. When compared to conventional cell-seeding, cell encapsulation within complex 3D patterned scaffolds provides long-term control over proliferation, cell morphology, and geometric guidance. Overall, this biofabrication technique offers a flexible platform to evaluate cell interactions with complex 3D micro-features, with the ability to scale-up towards high-throughput screening platforms. PMID:23686741

  2. Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

    Research highlights: → We irradiated a 3-D human oral cell culture of keratinocytes and fibroblasts with 12 and 2 Gy. → 6 h after irradiation the histopathology and apoptosis of the 3-D culture were evaluated. Microarrays were used to assess the gene expression in the irradiated 3-D tissue. → 12 Gy induced significant histopathologic changes and cellular apoptosis. → 12 Gy significantly affected genes of the NF-kB pathway, inflammatory cytokines and DAMPs. -- Abstract: Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first

  3. Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

    Lambros, Maria Polikandritou, E-mail: mlambros@westernu.edu [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Parsa, Cyrus [Department of Clinical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, Pomona, CA 91766 (United States); Mulamalla, HariChandana [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Orlando, Robert [Department of Clinical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, Pomona, CA 91766 (United States); Lau, Bernard [Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States); Huang, Ying [Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States); Pon, Doreen [Department of Pharmacy Practice and Administration, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Chow, Moses [Department of Pharmacy Practice and Administration, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766 (United States); Center for Advancement of Drug Research and Evaluation (CADRE), Western University of Health Sciences, Pomona, CA 91766 (United States)

    2011-02-04

    Research highlights: {yields} We irradiated a 3-D human oral cell culture of keratinocytes and fibroblasts with 12 and 2 Gy. {yields} 6 h after irradiation the histopathology and apoptosis of the 3-D culture were evaluated. Microarrays were used to assess the gene expression in the irradiated 3-D tissue. {yields} 12 Gy induced significant histopathologic changes and cellular apoptosis. {yields} 12 Gy significantly affected genes of the NF-kB pathway, inflammatory cytokines and DAMPs. -- Abstract: Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this

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

    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

  5. 3D porous biomimetically modified hydrogels supporting stem cells adhesion

    Studenovská, Hana; Vodička, Petr; Proks, Vladimír; Juhásová, Jana; Motlík, Jan; Rypáček, František

    Dublin : National University of Ireland , 2011. s. 119, psiii-630. [Annual Conference of the European Society for Biomaterials /24./. 04.09.2011-08.09.2011, Dublin] R&D Projects: GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50450515 Keywords : porous hydrogel * cell adhesion * polypeptide Subject RIV: FH - Neurology

  6. Expanding intestinal stem cells in culture

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  7. 3D-Modelling of polycrystalline silicon solar cells

    Dugas, J.; Oualid, J.

    1987-01-01

    This work is concerned with the effect of grain boundaries (gb) on the main parameters which characterize polycrystalline silicon solar cells. The variation with illumination of the grain boundary effective recombination velocity has been calculated by means of a self-consistent procedure which takes into account the bending of the minority carrier quasi-Fermi level in the space-charge region and in the gb quasi-neutral region. Abaci have been plotted which allow to predict the photovoltaic p...

  8. Rapid Assembly of Heterogeneous 3D Cell Microenvironments in a Microgel Array.

    Li, Yiwei; Chen, Pu; Wang, Yachao; Yan, Shuangqian; Feng, Xiaojun; Du, Wei; Koehler, Stephan A; Demirci, Utkan; Liu, Bi-Feng

    2016-05-01

    Heterogeneous 3D cell microenvironment arrays are rapidly assembled by combining surface-wettability-guided assembly and microdroplet-array-based operations. This approach enables precise control over individual shapes, sizes, chemical concentrations, cell density, and 3D spatial distribution of multiple components. This technique provides a cost-effective solution to meet the increasing demand of stem cell research, tissue engineering, and drug screening. PMID:26991071

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

    Carolyn M Higuchi

    Full Text Available 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

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

    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.

  11. A miniature microbial fuel cell with conducting nanofibers-based 3D porous biofilm

    Jiang, Huawei; Halverson, Larry J.; Dong, Liang

    2015-12-01

    Miniature microbial fuel cell (MFC) technology has received growing interest due to its potential applications in high-throughput screening of bacteria and mutants to elucidate mechanisms of electricity generation. This paper reports a novel miniature MFC with an improved output power density and short startup time, utilizing electrospun conducting poly(3,4-ethylenedioxythiophene) (PEDOT) nanofibers as a 3D porous anode within a 12 μl anolyte chamber. This device results in 423 μW cm-3 power density based on the volume of the anolyte chamber, using Shewanella oneidensis MR-1 as a model biocatalyst without any optimization of bacterial culture. The device also excels in a startup time of only 1hr. The high conductivity of the electrospun nanofibers makes them suitable for efficient electron transfer. The mean pore size of the conducting nanofibers is several micrometers, which is favorable for bacterial penetration and colonization of surfaces of the nanofibers. We demonstrate that S. oneidensis can fully colonize the interior region of this nanofibers-based porous anode. This work represents a new attempt to explore the use of electrospun PEDOT nanofibers as a 3D anode material for MFCs. The presented miniature MFC potentially will provide a high-sensitivity, high-throughput tool to screen suitable bacterial species and mutant strains for use in large-size MFCs.

  12. Towards the development of a 3D full cell and external busbars thermo-electric model

    Taking advantage of the increasing power of computers, it is now practical to consider building a 3D full cell and external busbars thermo-electric model. In the present study, a 3D full cell quarter thermo-electric model and a 3D cathode half plus liquid zone and busbars thermo-electric model have been developed and solved using a PIll 800 MHz computer. Developing a 3D full cell and external busbars thermo-electric model will constitute a step further towards the development of a fully 'multi-physic' unified aluminium reduction cell model. Already, a full cell thermo-electric model will be able to interact with a MHD model by providing it with accurate liquid zone current density and potshell temperature data and by receiving from it local liquid/ledge interface heat transfer coefficients. (author)

  13. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-06-01

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm). PMID:27219645

  14. Development of 3D in vitro platform technology to engineer mesenchymal stem cells

    Hosseinkhani H

    2012-06-01

    Full Text Available Hossein Hosseinkhani,1 Po-Da Hong,1 Dah-Shyong Yu,2 Yi-Ru Chen,3 Diana Ickowicz,4 Ira-Yudovin Farber,4 Abraham J Domb41Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (TAIWANTECH, 2Nanomedicine Research Center, National Defense Medical Center, Taipei, Taiwan, 3Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, 4Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, IsraelAbstract: This study aims to develop a three-dimensional in vitro culture system to genetically engineer mesenchymal stem cells (MSC to express bone morphogenic protein-2. We employed nanofabrication technologies borrowed from the spinning industry, such as electrospinning, to mass-produce identical building blocks in a variety of shapes and sizes to fabricate electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen. Homogenous nanoparticles of cationic biodegradable natural polymer were formed by simple mixing of an aqueous solution of plasmid DNA encoded bone morphogenic protein-2 with the same volume of cationic polysaccharide, dextran-spermine. Rat bone marrow MSC were cultured on electrospun nanofiber sheets comprised of composites of poly (glycolic acid and collagen prior to the incorporation of the nanoparticles into the nanofiber sheets. Bone morphogenic protein-2 was significantly detected in MSC cultured on nanofiber sheets incorporated with nanoparticles after 2 days compared with MSC cultured on nanofiber sheets incorporated with naked plasmid DNA. We conclude that the incorporation of nanoparticles into nanofiber sheets is a very promising strategy to genetically engineer MSC and can be used for further applications in regenerative medicine therapy.Keywords: 3D culture, nanoparticles, nanofibers, polycations, tissue engineering

  15. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals

    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. - Highlights: • 3D cultures using FGF-2 and FGF-7 microcrystals as a human skin model • Cytoprotection of keratinocytes against ROS by FGF-7 microcrystals • Overexpression of SOD and Prdx6 in keratinocytes by FGF-7 microcrystals

  16. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals

    Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-ichi [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Kotani, Eiji [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Kyoto (Japan); Hirano, Tomoko [Venture Laboratory, Kyoto Institute of Technology, Kyoto (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa (Japan); Matsumoto, Goichi [Division of Oral Surgery, Yokohama Clinical Education Center of Kanagawa Dental University, Yokohama (Japan); Mori, Hajime, E-mail: hmori@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Kyoto (Japan)

    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. - Highlights: • 3D cultures using FGF-2 and FGF-7 microcrystals as a human skin model • Cytoprotection of keratinocytes against ROS by FGF-7 microcrystals • Overexpression of SOD and Prdx6 in keratinocytes by FGF-7 microcrystals.

  17. Recovery of important physiological functions in 3D culture of immortal hepatocytes

    Wrzesinski, Krzysztof; Fey, S. J.

    2011-01-01

    grow human liver cells in ‘3 dimensional’ cultures so that they behave very similar to the liver in our bodies. By growing the immortal hepatocytes in specially designed bioreactors they form small pieces of ‘pseudotissue’ which exhibit several of the functions seen in the adult liver. We have grown...

  18. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming.

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-05-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  19. Cell Proliferation on Macro/Nano Surface Structure and Collagen Immobilization of 3D Polycaprolactone Scaffolds.

    Park, Young-Ouk; Myung, Sung-Woon; Kook, Min-Suk; Jung, Sang-Chul; Kim, Byung-Hoon

    2016-02-01

    In this study, 3D polycaprolactone (PCL) scaffolds were fabricated by 3D printing technique. The macro/nano morphology of, 3D PCL scaffolds surface was etched with oxygen plasma. Acrylic acid (AA) plasma-polymerization was performed to functionalize the macro/nano surface with carboxyl groups and then collagen was immobilized with plasma-polymerized 3D PCL scaffolds. After O2 plasma and AA plasma-polymerization, contact angles were decreased. The FE-SEM and AFM results showed that O2 plasma is increased the surface roughness. The MTT assay results showed that proliferation of the M3CT3-E1 cells increased on the oxygen plasma treated and collagen immobilized 3D PCL scaffolds. PMID:27433597

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

    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

  1. Micromorph silicon tandem solar cells with fully integrated 3D photonic crystal intermediate reflectors

    Üpping, J.; Bielawny, A.; Fahr, S.; Rockstuhl, C.; Lederer, F.; Steidl, L.; Zentel, R.; Beckers, T.; Lambertz, A.; Carius, R.; Wehrspohn, R. B.

    2010-05-01

    A 3D photonic intermediate reflector for textured micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell providing an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally-selective intermediate reflective layer (IRL) is necessary. We present the first fully-integrated 3D photonic thin-film IRL device incorporated on a planar substrate. Using a ZnO inverted opal structure the external quantum efficiency of the top cell in the spectral region of interest could be enhanced. As an outlook we present the design and the preparation of a 3D self organized photonic crystal structure in a textured micromorph tandem solar cell.

  2. 3D photonic crystal interlayers for micromorph thin film silicon tandem cell

    Uepping, Johannes; Bielawny, Andreas; Otto, Martin; Wehrspohn, Ralf B. [Institute of Physics, University of Halle, Wittenberg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, University of Mainz (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Halle (Germany); Beckers, Thomas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH (Germany)

    2010-07-01

    A 3D photonic intermediate reflector for textured micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/{mu}c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell. It is one goal to provide an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally selective intermediate reflective layer (IRL) is necessary. We show results toward the first fully integrated 3D photonic thin-film IRL device incorporated in a state-of-the-art textured tandem solar cell. The design and the preparation of a 3D self organized inverted opal photonic crystal structure in a textured micromorph tandem solar cell is presented.

  3. The Effects of Matrix Stiffness and RhoA on the Phenotypic Plasticity of Smooth Muscle Cells in a 3-D Biosynthetic Hydrogel System

    Peyton, Shelly R.; Kim, Peter D.; Ghajar, Cyrus M.; Seliktar, Dror; Putnam, Andrew J.

    2008-01-01

    Studies using 2-D cultures have shown that the mechanical properties of the extracellular matrix (ECM) influence cell migration, spreading, proliferation, and differentiation; however, cellular mechanosensing in 3-D remains under-explored. To investigate this topic, a unique biomaterial system based on poly(ethylene glycol)-conjugated fibrinogen was adapted to study phenotypic plasticity in smooth muscle cells (SMCs) as a function of ECM mechanics in 3-D. Tuning compressive modulus between 44...

  4. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

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

    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. Label-free characterization of white blood cells by measuring 3D refractive index maps

    Yoon, Jonghee; Park, HyunJoo; Choi, Chulhee; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs.

  7. High sensitivity plasmonic biosensor based on nanoimprinted quasi 3D nanosquares for cell detection.

    Zhu, Shuyan; Li, Hualin; Yang, Mengsu; Pang, Stella W

    2016-07-22

    Quasi three-dimensional (3D) plasmonic nanostructures consisting of Au nanosquares on top of SU-8 nanopillars and Au nanoholes on the bottom were developed and fabricated using nanoimprint lithography with simultaneous thermal and UV exposure. These 3D plasmonic nanostructures were used to detect cell concentration of lung cancer A549 cells, retinal pigment epithelial (RPE) cells, and breast cancer MCF-7 cells. Nanoimprint technology has the advantage of producing high uniformity plasmonic nanostructures for such biosensors. Multiple resonance modes were observed in these quasi 3D plasmonic nanostructures. The hybrid coupling of localized surface plasmon resonances and Fabry-Perot cavity modes in the quasi 3D nanostructures resulted in high sensitivity of 496 nm/refractive index unit. The plasmonic resonance peak wavelength and sensitivity could be tuned by varying the Au thickness. Resonance peak shifts for different cells at the same concentration were distinct due to their different cell area and confluency. The cell concentration detection limit covered a large range of 5 × 10(2) to 1 × 10(7) cells ml(-1) with these new plasmonic nanostructures. They also provide a large resonance peak shift of 51 nm for as little as 0.08 cells mm(-2) of RPE cells for high sensitivity cell detection. PMID:27275952

  8. High sensitivity plasmonic biosensor based on nanoimprinted quasi 3D nanosquares for cell detection

    Zhu, Shuyan; Li, Hualin; Yang, Mengsu; Pang, Stella W.

    2016-07-01

    Quasi three-dimensional (3D) plasmonic nanostructures consisting of Au nanosquares on top of SU-8 nanopillars and Au nanoholes on the bottom were developed and fabricated using nanoimprint lithography with simultaneous thermal and UV exposure. These 3D plasmonic nanostructures were used to detect cell concentration of lung cancer A549 cells, retinal pigment epithelial (RPE) cells, and breast cancer MCF-7 cells. Nanoimprint technology has the advantage of producing high uniformity plasmonic nanostructures for such biosensors. Multiple resonance modes were observed in these quasi 3D plasmonic nanostructures. The hybrid coupling of localized surface plasmon resonances and Fabry–Perot cavity modes in the quasi 3D nanostructures resulted in high sensitivity of 496 nm/refractive index unit. The plasmonic resonance peak wavelength and sensitivity could be tuned by varying the Au thickness. Resonance peak shifts for different cells at the same concentration were distinct due to their different cell area and confluency. The cell concentration detection limit covered a large range of 5 × 102 to 1 × 107 cells ml‑1 with these new plasmonic nanostructures. They also provide a large resonance peak shift of 51 nm for as little as 0.08 cells mm‑2 of RPE cells for high sensitivity cell detection.

  9. MULTILEVEL (3D) MICROFLUIDIC TECHNOLOGY FOR AN INNOVATIVE MAGNETIC CELL SEPARATION PLATFORM

    Fouet, Marc; Cargou, Sébastien; Courson, Rémi; Blatché, Charline; Montrose, A.; Reybier, K; Gué, Anne-Marie

    2014-01-01

    We demonstrate a new concept of devices, which by combining 3D fluid engineering and localized mag-netic actuation enables the full integration of a cell tagging and magnetic separation device. We used a low cost, commercially available dry film (EMS Inc, Ohio, USA) that fits microfluidic requirements and gives the possibility to build easily 3D microfluidic structures. The labelling of blood monocytes with su-perparamagnetic particles was performed "up stream" with the aim of a microparticle...

  10. Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids.

    Beaumont, Kimberley A; Anfosso, Andrea; Ahmed, Farzana; Weninger, Wolfgang; Haass, Nikolas K

    2015-01-01

    Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. PMID:26779761

  11. Heritable Genetic Changes in Cells Recovered From Irradiated 3D Tissue Contracts. Final report

    Cornforth, Michael N. [The University of Texas Medical Branch at Galveston, TX (United States)

    2013-05-03

    Combining contemporary cytogenetic methods with DNA CGH microarray technology and chromosome flow-sorting increases substantially the ability to resolve exchange breakpoints associated with interstitial deletions and translocations, allowing the consequences of radiation damage to be directly measured at low doses, while also providing valuable insights into molecular mechanisms of misrepair processes that, in turn, identify appropriate biophysical models of risk at low doses. The aims of this work apply to cells recovered from 3D tissue constructs of human skin and, for the purpose of comparison, the same cells irradiated in traditional 2D cultures. These aims are: to analyze by multi-flour fluorescence in situ hybridization (mFISH) the chromosomes in clonal descendents of individual human fibroblasts that were previously irradiated; to examine irradiated clones from Aim 1 for submicroscopic deletions by subjecting their DNA to comparative genomic hybridization (CGH) microarray analysis; and to flow-sort aberrant chromosomes from clones containing stable radiation-induced translocations and map the breakpoints to within an average resolution of 100 kb using the technique of 'array painting'.

  12. Tuning Cell Differentiation into a 3D Scaffold Presenting a Pore Shape Gradient for Osteochondral Regeneration.

    Di Luca, Andrea; Lorenzo-Moldero, Ivan; Mota, Carlos; Lepedda, Antonio; Auhl, Dietmar; Van Blitterswijk, Clemens; Moroni, Lorenzo

    2016-07-01

    Osteochondral regeneration remains nowadays a major problem since the outcome of current techniques is not satisfactory in terms of functional tissue formation and development. A possible solution is the combination of human mesenchymal stem cells (hMSCs) with additive manufacturing technologies to fabricate scaffolds with instructive properties. In this study, the differentiation of hMSCs within a scaffold presenting a gradient in pore shape is presented. The variation in pore shape is determined by varying the angle formed by the fibers of two consequent layers. The fiber deposition patterns are 0-90, which generate squared pores, 0-45, 0-30, and 0-15, that generate rhomboidal pores with an increasing major axis as the deposition angle decreases. Within the gradient construct, squared pores support a better chondrogenic differentiation whereas cells residing in the rhomboidal pores display a better osteogenic differentiation. When cultured under osteochondral conditions the trend in both osteogenic and chondrogenic markers is maintained. Engineering the pore shape, thus creating axial gradients in structural properties, seems to be an instructive strategy to fabricate functional 3D scaffolds that are able to influence hMSCs differentiation for osteochondral tissue regeneration. PMID:27109461

  13. Heritable Genetic Changes in Cells Recovered From Irradiated 3D Tissue Contracts. Final report

    Combining contemporary cytogenetic methods with DNA CGH microarray technology and chromosome flow-sorting increases substantially the ability to resolve exchange breakpoints associated with interstitial deletions and translocations, allowing the consequences of radiation damage to be directly measured at low doses, while also providing valuable insights into molecular mechanisms of misrepair processes that, in turn, identify appropriate biophysical models of risk at low doses. The aims of this work apply to cells recovered from 3D tissue constructs of human skin and, for the purpose of comparison, the same cells irradiated in traditional 2D cultures. These aims are: to analyze by multi-flour fluorescence in situ hybridization (mFISH) the chromosomes in clonal descendents of individual human fibroblasts that were previously irradiated; to examine irradiated clones from Aim 1 for submicroscopic deletions by subjecting their DNA to comparative genomic hybridization (CGH) microarray analysis; and to flow-sort aberrant chromosomes from clones containing stable radiation-induced translocations and map the breakpoints to within an average resolution of 100 kb using the technique of 'array painting'

  14. BMP4 inhibits the proliferation of breast cancer cells and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells in 3D environment

    Bone morphogenetic protein 4 (BMP4) belongs to the transforming growth factor β (TGF-β) family of proteins. BMPs regulate cell proliferation, differentiation and motility, and have also been reported to be involved in cancer pathogenesis. We have previously shown that BMP4 reduces breast cancer cell proliferation through G1 cell cycle arrest and simultaneously induces migration in a subset of these cell lines. Here we examined the effects of BMP4 in a more physiological environment, in a 3D culture system. We used two different 3D culture systems; Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel. AlamarBlue reagent was used for cell proliferation measurements and immunofluorescence was used to determine cell polarity. Expression of cell cycle regulators was examined by Western blot and matrix metalloproteinase (MMP) expression by qRT-PCR. The MCF-10A normal breast epithelial cells formed round acini with correct apicobasal localization of α6 integrin in Matrigel whereas irregular structures were seen in PEG gel. The two 3D matrices also supported dissimilar morphology for the breast cancer cells. In PEG gel, BMP4 inhibited the growth of MCF-10A and the three breast cancer cell lines examined, thus closely resembling the 2D culture conditions, but in Matrigel, no growth inhibition was observed in MDA-MB-231 and MDA-MB-361 cells. Furthermore, BMP4 induced the expression of the cell cycle inhibitor p21 both in 2D and 3D culture, thereby partly explaining the growth arrest. Interestingly, MDA-MB-231 cells formed large branching, stellate structures in response to BMP4 treatment in Matrigel, suggestive of increased cell migration or invasion. This effect was reversed by Batimastat, a broad-spectrum MMP inhibitor, and subsequent analyses showed BMP4 to induce the expression of MMP3 and MMP14, that are thus likely to be responsible for the stellate phenotype. Taken together, our results show that Matrigel

  15. 3D photonic crystal interlayers for micromorph thin film silicon tandem cell

    Bielawny, Andreas; Uepping, Johannes; Miclea, Paul T.; Wehrspohn, Ralf B. [Institute of Physics, University of Halle, Wittenberg (Germany); Rockstuhl, Carsten; Lederer, Falk [Institue of Physics, Solid States Optics, University of Jena (Germany); Peters, Marius [Freiburg Centre for Material Research, University of Freiburg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, Pharmacy and Earth Science, University of Mainz (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Halle (Germany); Lambertz, Andreas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH (Germany)

    2009-07-01

    The concept of 3D photonic intermediate reflectors for micromorph silicon tandem cells has been investigated toward first prototype cells. The reflector enhances the absorption of spectrally selected light in the top cell and decreases the current mismatch between both junctions. Our device is an inverted opal structure made of ZnO and built using self organized nanoparticles and atomic layer deposition coating methods. This 3D photonic crystal intermediate layer is less dependent of the angle of incidence than other state of the art thickness dependent massive interlayers. We present design rules, preparation and characterization of a 3D photonic thin film device. A first prototype is compared to a state of the art reference silicon tandem cell.

  16. Cell volume and geometric parameters determination in living cells using confocal microscopy and 3D reconstruction

    sprotocols

    2015-01-01

    Authors: David Hevia, Aida Rodriguez-Garcia, Marta Alonso-Gervós, Isabel Quirós-González, Henar M Cimadevilla, Carmen Gómez-Cordovés, Rosa M Sainz & Juan C Mayo ### Abstract The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO...

  17. 3D-printed concentrator arrays for external light trapping on thin film solar cells

    van Dijk, Lourens; Marcus, E. A. Pepijn; Oostra, A. Jolt; Schropp, Ruud E. I.; Di Vece, Marcel

    2015-01-01

    After our recent demonstration of a 3D-printed external light trap on a small solar cell, we now consider its potential for large solar panels. An external light trap consists of a parabolic concentrator and a spacer that redirects the photons that are reflected by the solar cell back towards the so

  18. 3D Image-Guided Automatic Pipette Positioning for Single Cell Experiments in vivo

    Brian Long; Lu Li; Ulf Knoblich; Hongkui Zeng; Hanchuan Peng

    2015-01-01

    We report a method to facilitate single cell, image-guided experiments including in vivo electrophysiology and electroporation. Our method combines 3D image data acquisition, visualization and on-line image analysis with precise control of physical probes such as electrophysiology microelectrodes in brain tissue in vivo. Adaptive pipette positioning provides a platform for future advances in automated, single cell in vivo experiments.

  19. A 3D in vitro model reveals differences in the astrocyte response elicited by potential stem cell therapies for CNS injury

    East, Emma; Johns, Noemie; Georgiou, Melanie; Golding, Jon; Loughlin, Jane; Kingham, Paul; Phillips, James

    2013-01-01

    Aim: This study aimed to develop a 3D culture model to test the extent to which transplanted stem cells modulate astrocyte reactivity, where exacerbated glial cell activation could be detrimental to CNS repair success. Materials & methods: The reactivity of rat astrocytes to bone marrow mesenchymal stem cells, neural crest stem cells (NCSCs) and differentiated adipose-derived stem cells was assessed after 5 days. Schwann cells were used as a positive control. Results: NCSCs and differ...

  20. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-06-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

  1. From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy.

    Spiegelhalter, Coralie; Tosch, Valérie; Hentsch, Didier; Koch, Marc; Kessler, Pascal; Schwab, Yannick; Laporte, Jocelyn

    2010-01-01

    BACKGROUND: In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). METHODOLOGY: To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coo...

  2. Digital Image Analysis of Cells : Applications in 2D, 3D and Time

    Pinidiyaarachchi, Amalka

    2009-01-01

    Light microscopes are essential research tools in biology and medicine. Cell and tissue staining methods have improved immensely over the years and microscopes are now equipped with digital image acquisition capabilities. The image data produced require development of specialized analysis methods. This thesis presents digital image analysis methods for cell image data in 2D, 3D and time sequences. Stem cells have the capability to differentiate into specific cell types. The mechanism behind di...

  3. An Innovative Cell Microincubator for Drug Discovery Based on 3D Silicon Structures

    Francesca Aredia

    2016-01-01

    Full Text Available We recently employed three-dimensional (3D silicon microstructures (SMSs consisting in arrays of 3 μm-thick silicon walls separated by 50 μm-deep, 5 μm-wide gaps, as microincubators for monitoring the biomechanical properties of tumor cells. They were here applied to investigate the in vitro behavior of HT1080 human fibrosarcoma cells driven to apoptosis by the chemotherapeutic drug Bleomycin. Our results, obtained by fluorescence microscopy, demonstrated that HT1080 cells exhibited a great ability to colonize the narrow gaps. Remarkably, HT1080 cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and were prone to leave the gaps. Finally, we performed label-free detection of cells adherent to the vertical silicon wall, inside the gap of 3D-SMS, by exploiting optical low coherence reflectometry using infrared, low power radiation. This kind of approach may become a new tool for increasing automation in the drug discovery area. Our results open new perspectives in view of future applications of the 3D-SMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells.

  4. Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites

    Jung, Jangwook P.; Bache-Wiig, Meredith K.; Provenzano, Paolo P.; Ogle, Brenda M.

    2016-01-01

    Abstract Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression

  5. A new method to address unmet needs for extracting individual cell migration features from a large number of cells embedded in 3D volumes.

    Ivan Adanja

    Full Text Available BACKGROUND: In vitro cell observation has been widely used by biologists and pharmacologists for screening molecule-induced effects on cancer cells. Computer-assisted time-lapse microscopy enables automated live cell imaging in vitro, enabling cell behavior characterization through image analysis, in particular regarding cell migration. In this context, 3D cell assays in transparent matrix gels have been developed to provide more realistic in vitro 3D environments for monitoring cell migration (fundamentally different from cell motility behavior observed in 2D, which is related to the spread of cancer and metastases. METHODOLOGY/PRINCIPAL FINDINGS: In this paper we propose an improved automated tracking method that is designed to robustly and individually follow a large number of unlabeled cells observed under phase-contrast microscopy in 3D gels. The method automatically detects and tracks individual cells across a sequence of acquired volumes, using a template matching filtering method that in turn allows for robust detection and mean-shift tracking. The robustness of the method results from detecting and managing the cases where two cell (mean-shift trackers converge to the same point. The resulting trajectories quantify cell migration through statistical analysis of 3D trajectory descriptors. We manually validated the method and observed efficient cell detection and a low tracking error rate (6%. We also applied the method in a real biological experiment where the pro-migratory effects of hyaluronic acid (HA were analyzed on brain cancer cells. Using collagen gels with increased HA proportions, we were able to evidence a dose-response effect on cell migration abilities. CONCLUSIONS/SIGNIFICANCE: The developed method enables biomedical researchers to automatically and robustly quantify the pro- or anti-migratory effects of different experimental conditions on unlabeled cell cultures in a 3D environment.

  6. 5D Modelling: An Efficient Approach for Creating Spatiotemporal Predictive 3D Maps of Large-Scale Cultural Resources

    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

  7. Study of a Microfluidic Chip Integrating Single Cell Trap and 3D Stable Rotation Manipulation

    Liang Huang

    2016-08-01

    Full Text Available Single cell manipulation technology has been widely applied in biological fields, such as cell injection/enucleation, cell physiological measurement, and cell imaging. Recently, a biochip platform with a novel configuration of electrodes for cell 3D rotation has been successfully developed by generating rotating electric fields. However, the rotation platform still has two major shortcomings that need to be improved. The primary problem is that there is no on-chip module to facilitate the placement of a single cell into the rotation chamber, which causes very low efficiency in experiment to manually pipette single 10-micron-scale cells into rotation position. Secondly, the cell in the chamber may suffer from unstable rotation, which includes gravity-induced sinking down to the chamber bottom or electric-force-induced on-plane movement. To solve the two problems, in this paper we propose a new microfluidic chip with manipulation capabilities of single cell trap and single cell 3D stable rotation, both on one chip. The new microfluidic chip consists of two parts. The top capture part is based on the least flow resistance principle and is used to capture a single cell and to transport it to the rotation chamber. The bottom rotation part is based on dielectrophoresis (DEP and is used to 3D rotate the single cell in the rotation chamber with enhanced stability. The two parts are aligned and bonded together to form closed channels for microfluidic handling. Using COMSOL simulation and preliminary experiments, we have verified, in principle, the concept of on-chip single cell traps and 3D stable rotation, and identified key parameters for chip structures, microfluidic handling, and electrode configurations. The work has laid a solid foundation for on-going chip fabrication and experiment validation.

  8. HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment

    Weigelt, Britta; Lo, Alvin T; Park, Catherine C; Gray, Joe W; Bissell, Mina J

    2009-07-27

    Development of effective and durable breast cancer treatment strategies requires a mechanistic understanding of the influence of the microenvironment on response. Previous work has shown that cellular signaling pathways and cell morphology are dramatically influenced by three-dimensional (3D) cultures as opposed to traditional two-dimensional (2D) monolayers. Here, we compared 2D and 3D culture models to determine the impact of 3D architecture and extracellular matrix (ECM) on HER2 signaling and on the response of HER2-amplified breast cancer cell lines to the HER2-targeting agents Trastuzumab, Pertuzumab and Lapatinib. We show that the response of the HER2-amplified AU565, SKBR3 and HCC1569 cells to these anti-HER2 agents was highly dependent on whether the cells were cultured in 2D monolayer or 3D laminin-rich ECM gels. Inhibition of {beta}1 integrin, a major cell-ECM receptor subunit, significantly increased the sensitivity of the HER2-amplified breast cancer cell lines to the humanized monoclonal antibodies Trastuzumab and Pertuzumab when grown in a 3D environment. Finally, in the absence of inhibitors, 3D cultures had substantial impact on HER2 downstream signaling and induced a switch between PI3K-AKT- and RAS-MAPKpathway activation in all cell lines studied, including cells lacking HER2 amplification and overexpression. Our data provide direct evidence that breast cancer cells are able to rapidly adapt to different environments and signaling cues by activating alternative pathways that regulate proliferation and cell survival, events that may play a significant role in the acquisition of resistance to targeted therapies.

  9. Mechano-sensing and cell migration: a 3D model approach

    Cell migration is essential for tissue development in different physiological and pathological conditions. It is a complex process orchestrated by chemistry, biological factors, microstructure and surrounding mechanical properties. Focusing on the mechanical interactions, cells do not only exert forces on the matrix that surrounds them, but they also sense and react to mechanical cues in a process called mechano-sensing. Here, we hypothesize the involvement of mechano-sensing in the regulation of directional cell migration through a three-dimensional (3D) matrix. For this purpose, we develop a 3D numerical model of individual cell migration, which incorporates the mechano-sensing process of the cell as the main mechanism regulating its movement. Consistent with this hypothesis, we found that factors, such as substrate stiffness, boundary conditions and external forces, regulate specific and distinct cell movements

  10. 3D Culture of Chondrocytes in Gelatin Hydrogels with Different Stiffness

    Xiaomeng Li

    2016-07-01

    Full Text Available Gelatin hydrogels can mimic the microenvironments of natural tissues and encapsulate cells homogeneously, which makes them attractive for cartilage tissue engineering. Both the mechanical and biochemical properties of hydrogels can affect the phenotype of chondrocytes. However, the influence of each property on chondrocyte phenotype is unclear due to the difficulty in separating the roles of these properties. In this study, we aimed to study the influence of hydrogel stiffness on chondrocyte phenotype while excluding the role of biochemical factors, such as adhesion site density in the hydrogels. By altering the degree of methacryloyl functionalization, gelatin hydrogels with different stiffnesses of 3.8, 17.1, and 29.9 kPa Young’s modulus were prepared from the same concentration of gelatin methacryloyl (GelMA macromers. Bovine articular chondrocytes were encapsulated in the hydrogels and cultured for 14 days. The influence of hydrogel stiffness on the cell behaviors including cell viability, cell morphology, and maintenance of chondrogenic phenotype was evaluated. GelMA hydrogels with high stiffness (29.9 kPa showed the best results on maintaining chondrogenic phenotype. These results will be useful for the design and preparation of scaffolds for cartilage tissue engineering.

  11. Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

    Paris Jafari

    Full Text Available Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i 3-OHGA causes the death of astrocytes, (ii deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

  12. 3D staggered Lagrangian hydrodynamics scheme with cell-centered Riemann solver-based artificial viscosity

    The aim of the present work is the 3D extension of a general formalism to derive a staggered discretization for Lagrangian hydrodynamics on unstructured grids. The classical compatible discretization is used; namely, momentum equation is discretized using the fundamental concept of subcell forces. Specific internal energy equation is obtained using total energy conservation. The subcell force is derived by invoking the Galilean invariance and thermodynamic consistency. A general form of the subcell force is provided so that a cell entropy inequality is satisfied. The subcell force consists of a classical pressure term plus a tensorial viscous contribution proportional to the difference between the node velocity and the cell-centered velocity. This cell-centered velocity is an extra degree of freedom solved with a cell-centered approximate Riemann solver. The second law of thermodynamics is satisfied by construction of the local positive definite subcell tensor involved in the viscous term. A particular expression of this tensor is proposed. A more accurate extension of this discretization both in time and space is also provided using a piecewise linear reconstruction of the velocity field and a predictor-corrector time discretization. Numerical tests are presented in order to assess the efficiency of this approach in 3D. Sanity checks show that the 3D extension of the 2D approach reproduces 1D and 2D results. Finally, 3D problems such as Sedov, Noh, and Saltzman are simulated. (authors)

  13. A 3D GCL compatible cell-centered Lagrangian scheme for solving gas dynamics equations

    Georges, Gabriel; Breil, Jérôme; Maire, Pierre-Henri

    2016-01-01

    Solving the gas dynamics equations under the Lagrangian formalism enables to simulate complex flows with strong shock waves. This formulation is well suited to the simulation of multi-material compressible fluid flows such as those encountered in the domain of High Energy Density Physics (HEDP). These types of flows are characterized by complex 3D structures such as hydrodynamic instabilities (Richtmyer-Meshkov, Rayleigh-Taylor, etc.). Recently, the 3D extension of different Lagrangian schemes has been proposed and appears to be challenging. More precisely, the definition of the cell geometry in the 3D space through the treatment of its non-planar faces and the limiting of a reconstructed field in 3D in the case of a second-order extension are of great interest. This paper proposes two new methods to solve these problems. A systematic and symmetric geometrical decomposition of polyhedral cells is presented. This method enables to define a discrete divergence operator leading to the respect of the Geometric Conservation Law (GCL). Moreover, a multi-dimensional minmod limiter is proposed. This new limiter constructs, from nodal gradients, a cell gradient which enables to ensure the monotonicity of the numerical solution even in presence of strong discontinuity. These new ingredients are employed into a cell-centered Lagrangian scheme. Robustness and accuracy are assessed against various representative test cases.

  14. Correlative 3D imaging of Whole Mammalian Cells with Light and Electron Microscopy

    Murphy, Gavin E.; Narayan, Kedar; Lowekamp, Bradley C.; Hartnell, Lisa M.; Heymann, Jurgen A. W.; Fu, Jing; Subramaniam, Sriram

    2011-01-01

    We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA–SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ~10 to 20 nm at high contrast and with minimal artifacts from the foc...

  15. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

    Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan; Wolff, Anders; Emnéus, Jenny; Aspegren, Anders; Dufva, Martin

    A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due to...... differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....

  16. Quantitative data analysis methods for 3D microstructure characterization of Solid Oxide Cells

    Jørgensen, Peter Stanley

    . Alignment of the individual image slices is performed by automatic detection of ducial marks. Uneven illumination is corrected by tting hypersurfaces to the spatial intensity variation in the 3D image data. Routine use of quantitative three dimensional analysis of microstructure is generally restricted by...... for gaining further fundamental understanding of how microstructure affects performance. In this work, methods for automatic 3D characterization of microstructure are studied: from the acquisition of 3D image data by focused ion beam tomography to the extraction of quantitative measures that......The performance of electrochemical ceramic devices such as solid oxide fuel and electrolyser cells depends on the distribution of constituent phases on the micro or nano scale, also known as the microstructure. The microstructure governs key properties such as ion, electron and gas transport...

  17. 3D Visualization of Cultural Heritage Artefacts with Virtual Reality devices

    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.

  18. 3D Realisation of Cultural Heritage UNESCO world heritage site in Saltaire, Bradford

    Unver, Ertu; Taylor, Andrew

    2010-01-01

    This presentation is given at the Virtual Heritage 2010, Bangor, UK. The team is invited to present the progress of the current research & development in the 3D Digital Heritage visualisation project based on UNESCO world Heritage site at Saltaire in West Yorkshire UK.

  19. Uncovering cancer cell behavioral phenotype in 3-D in vitro metastatic landscapes

    Liu, Liyu; Sun, Bo; Duclos, Guillaume; Kam, Yoonseok; Gatenby, Robert; Stone, Howard; Austin, Robert

    2012-02-01

    One well-known fact is that cancer cell genetics determines cell metastatic potentials. However, from a physics point of view, genetics as cell properties cannot directly act on metastasis. An agent is needed to unscramble the genetics first before generating dynamics for metastasis. Exactly this agent is cell behavioral phenotype, which is rarely studied due to the difficulties of real-time cell tracking in in vivo tissue. Here we have successfully constructed a micro in vitro environment with collagen based Extracellular Matrix (ECM) structures for cell 3-D metastasis. With stable nutrition (glucose) gradient inside, breast cancer cell MDA-MB-231 is able to invade inside the collagen from the nutrition poor site towards the nutrition rich site. Continuous confocal microscopy captures images of the cells every 12 hours and tracks their positions in 3-D space. The micro fluorescent beads pre-mixed inside the ECM demonstrate that invasive cells have altered the structures through mechanics. With the observation and the analysis of cell collective behaviors, we argue that game theory may exist between the pioneering cells and their followers in the metastatic cell group. The cell collaboration may explain the high efficiency of metastasis.

  20. Fabrication of Dye-Sensitized Solar Cells with a 3D Nanostructured Electrode

    Guo-Yang Chen

    2010-01-01

    Full Text Available A novel Dye-Sensitized Solar Cell (DSSC scheme for better solar conversion efficiency is proposed. The distinctive characteristic of this novel scheme is that the conventional thin film electrode is replaced by a 3D nanostructured indium tin oxide (ITO electrode, which was fabricated using RF magnetron sputtering with an anodic aluminum oxide (AAO template. The template was prepared by immersing the barrier-layer side of an AAO film into a 30 wt% phosphoric acid solution to produce a contrasting surface. RF magnetron sputtering was then used to deposit a 3D nanostructured ITO thin film on the template. The crystallinity and conductivity of the 3D ITO films were further enhanced by annealing. Titanium dioxide nanoparticles were electrophoretically deposited on the 3D ITO film after which the proposed DSSC was formed by filling vacant spaces in the 3D nanostructured ITO electrode with dye. The measured solar conversion efficiency of the device was 0.125%. It presents a 5-fold improvement over that of conventional spin-coated TiO2 film electrode DSSCs.

  1. Computational Graph Model for 3D Cells Tracking in Zebra Fish Datasets

    Zhang, Lelin; Xiong, Hongkai; Zhao, Yang; Zhang, Kai; Zhou, Xiaobo

    2007-11-01

    This paper leads to a novel technique for tracking and identification of zebra-fish cells in 3D image sequences, extending graph-based multi-objects tracking algorithm to 3D applications. As raised in previous work of 2D graph-based method, separated cells are modeled as vertices that connected by edges. Then the tracking work is simplified to that of vertices matching between graphs generated from consecutive frames. Graph-based tracking is composed of three steps: graph generation, initial source vertices selection and graph saturation. To satisfy demands in this work separated cell records are segmented from original datasets using 3D level-set algorithms. Besides, advancements are achieved in each of the step including graph regulations, multi restrictions on source vertices and enhanced flow quantifications. Those strategies make a good compensation for graph-based multi-objects tracking method in 2D space. Experiments are carried out in 3D datasets sampled from zebra fish, results of which shows that this enhanced method could be potentially applied to tracking of objects with diverse features.

  2. Organic MEMS/NEMS-based high-efficiency 3D ITO-less flexible photovoltaic cells

    A novel approach based on three-dimensional (3D) architecture for polymeric photovoltaic cells made up of an array of sub-micron and nano-pillars which not only increase the area of the light absorbing surface, but also improve the carrier collection efficiency of bulk-heterojunction organic solar cells is presented. The approach also introduces coating of 3D anodes with a new solution-processable highly conductive transparent polymer (Orgacon™) that replaces expensive vacuum-deposited ITO (indium tin oxide) as well as the additional hole-collecting layer of conventional PEDOT:PSS (poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate)). In addition, the described procedure is well suited to roll-to-roll high-throughput manufacturing. The high aspect-ratio 3D pillars which form the basis for this new architecture are patterned through micro-electromechanical-system- and nano-electromechanical-system-based processes. For the particular case of P3HT (poly(3-hexylthiophene)) and PCBM (phenyl-C61-butyric acid methyl ester) active material, efficiencies in excess of 6% have been achieved for these photovoltaic cells of 3D architecture using ITO-less flexible PET (polyethylene terephthalate) substrates. This increase in efficiency turns out to be more than twice higher than those achieved for their 2D counterparts. (paper)

  3. 3D-printing of Redox flow batteries for energy storage: a rapid prototype laboratory cell

    Arenas-Martinez, L.F.; Walsh, F.C.; Ponce de Leon, C.

    2015-01-01

    Although interest in redox flow batteries (RFBs) for energy storage has grown over the last few years, implementation of RFB technology has been slow and challenging. Recent developments in 3D-printing of materials enable a transforming technology for fast, reproducible and documented cell manufacture. This technology can give an improved engineering approach to cell design and fabrication, needed to fulfil requirements for lower cost, longer lifetime hardware capable of efficient reliable pe...

  4. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells

    Alain Carpentier; Juan C. Chachques; Fabien Legrand; Samira Benadda; Nermine Lila; Kanwal Haneef

    2012-01-01

    Electrostimulation (ES) can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs) seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 x 25 x 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical im...

  5. Cell counting in human endobronchial biopsies--disagreement of 2D versus 3D morphometry.

    Vlad A Bratu

    Full Text Available QUESTION: Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel. MATERIALS AND METHODS: Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7 and smokers (n = 7, embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68 and T-lymphocytes (CD3, respectively. In identical fields of view, cell numbers per volume unit (NV were assessed using the physical disector (3D, and profiles per area unit (NA were counted (2D. For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis. RESULTS: In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05. When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups. CONCLUSIONS: 2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a 'universal conversion formula'.

  6. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  7. Photopatterning of Hydrogel Scaffolds Coupled to Filter Materials Using Stereolithography for Perfused 3D Culture of Hepatocytes

    Shepard Neiman, Jaclyn A.; 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.

    2015-01-01

    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 fabricate...

  8. A 3-D Model of a Perennial Ryegrass Primary Cell Wall and Its Enzymatic Degradation

    Indrakumar Vetharaniam; Kelly, William J.; Graeme T. Attwood; Harris, Philip J.

    2014-01-01

    We have developed a novel 3-D, agent-based model of cell-wall digestion to improve our understanding of ruminal cell-wall digestion. It offers a capability to study cell walls and their enzymatic modification, by providing a representation of cellulose microfibrils and non-cellulosic polysaccharides and by simulating their spatial and catalytic interactions with enzymes. One can vary cell-wall composition and the types and numbers of enzyme molecules, allowing the model to be applied to a ran...

  9. Advances in 3D Spatial Information Systems. Applications in cultural heritage and virtual archeology

    María Dolores Robles Ortega

    2015-10-01

    Full Text Available Large point clouds from radars and these-dimensional scanners are commonly used in Archaeology. However, in most cases these models cannot be properly integrated and used in software such as heritage management due to its large size. Therefore, some tools to make this management easier and optimize the processing are needed. In this work, we propone the integration between OpenVDB and GRASS in a C++ module to combine the widen functionality of GRASS GIS with the 3D models management efficiency of OpenVDB. Specifically, this application is used to combine the topographic information of a city with the 3D models of the most significant buildings. This application can be useful for both current cities as well as for virtual reconstruction of existing villages in the olden days and currently disappeared.

  10. 3D Printing of Scaffold for Cells Delivery: Advances in Skin Tissue Engineering

    Deepti Singh

    2016-01-01

    Full Text Available Injury or damage to tissue and organs is a major health problem, resulting in about half of the world’s annual healthcare expenditure every year. Advances in the fields of stem cells (SCs and biomaterials processing have provided a tremendous leap for researchers to manipulate the dynamics between these two, and obtain a skin substitute that can completely heal the wounded areas. Although wound healing needs a coordinated interplay between cells, extracellular proteins and growth factors, the most important players in this process are the endogenous SCs, which activate the repair cascade by recruiting cells from different sites. Extra cellular matrix (ECM proteins are activated by these SCs, which in turn aid in cellular migrations and finally secretion of growth factors that can seal and heal the wounds. The interaction between ECM proteins and SCs helps the skin to sustain the rigors of everyday activity, and in an attempt to attain this level of functionality in artificial three-dimensional (3D constructs, tissue engineered biomaterials are fabricated using more advanced techniques such as bioprinting and laser assisted printing of the organs. This review provides a concise summary of the most recent advances that have been made in the area of polymer bio-fabrication using 3D bio printing used for encapsulating stem cells for skin regeneration. The focus of this review is to describe, in detail, the role of 3D architecture and arrangement of cells within this system that can heal wounds and aid in skin regeneration.

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

    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.

  12. 3D Visualization of Cultural Heritage Artefacts with Virtual Reality devices

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

    2015-01-01

    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....

  13. Virtual 3D museum of Neolithic culture. Artefacts, technology and imagining

    Květina, Petr; Brzobohatá, Hana; Burgert, Pavel; Končelová, Markéta; Pavlů, Ivan; Unger, Jiří; Řídký, Jaroslav; Šumberová, Radka; Vavrečka, Petr

    Istanbul: Archaeology & Art Publications, 2014 - (Yilmaz, Ö.). s. 205-206 ISBN 978-605-396-287-8. [Annual Meeting of the European Association of Archaeologists /20./. 10.09.2014-14.09.2014, Istanbul] R&D Projects: GA MK(CZ) DF12P01OVV032 Keywords : Neolithic * virtual museum * 3D * augmented reality Subject RIV: AC - Archeology, Anthropology, Ethnology https://www.eaa2014istanbul.org/assets/indirilecekler/2014%20EAA%20abstracts.pdf

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

    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...

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

    Hong Zhou

    Full Text Available 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

  16. 3D photonic crystal intermediate reflector for micromorph thin-film tandem solar cell

    Uepping, Johannes; Miclea, Paul T.; Wehrspohn, Ralf B. [Institute of Physics, Martin-Luther-University of Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle (Germany); Rockstuhl, Carsten; Lederer, Falk [Institute of Condensed Matter Theory and Solid States Optics, Friedrich Schiller University Jena, 07743 Jena (Germany); Peters, Marius [Freiburg Centre for Material Research, University of Freiburg, 79104 Freiburg (Germany); Steidl, Lorenz; Zentel, Rudolf [Dept. of Chemistry, Pharmacy and Earth Science, Johannes Gutenberg University of Mainz, Duesbergweg 10-14 (Germany); Lee, Seung-Mo; Knez, Mato [Max Planck Institute of Microstructure Physics, Weinberg 2, 06120 Halle (Germany); Lambertz, Andreas; Carius, Reinhard [Institute of Energy Research, IEF-5 Photovoltaics, Forschungszentrum Juelich GmbH, 52425 Juelich (Germany); Bielawny, Andreas

    2008-12-15

    The concept of 3D photonic intermediate reflectors for micromorph silicon tandem solar cells has been investigated. In thin-film silicon tandem solar cells consisting of amorphous and microcrystalline silicon with two junctions of a-Si/{mu}c-Si, efficiency enhancements can be achieved by increasing the current density in the a-Si top cell. It is one goal to provide an optimized current matching at high current densities. For an ideal photon-management between top and bottom cell, a spectrally selective intermediate reflective layer (IRL) is necessary, which is less dependent of the angle of incidence than state-of-the-art thickness dependent massive interlayers. The design, preparation and characterization of a 3D photonic thin-film filter device for this purpose has been pursued straight forward in simulation and experimental realization. The inverted opal is capable of providing a suitable optical band stop with high reflectance and the necessary long wavelength transmittance as well and provides further options for improved light trapping. We have determined numerically the relative efficiency enhancement of an a-Si/{mu}c-Si tandem solar cell using a conductive 3D-photonic crystal. We have further fabricated such structures by ZnO-replication of polymeric opals using chemical vapour deposition and atomic layer deposition techniques and present the results of their characterization. Thin film photonic IRL have been prepared at the rear side of a-Si solar cells. Completed with a back contact, this is the first step to integrate this novel technology into an a-Si/{mu}c-Si tandem solar cell process. The spectral response of the cell is presented and compared with reference cells. (copyright 2008 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

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

    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

  18. Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells

    Roma, P M S; Amaral, F T; Agero, U; Mesquita, O N

    2014-01-01

    We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 3D imaging of transparent objects. By total 3D imaging we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition.

  19. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy

    Park, HyunJoo; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, SangEun; Park, YongKeun

    2015-01-01

    Babesia microti causes emergency human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.

  20. Determination of key parameters of SEU occurrence using 3-D full cell SRAM simulations

    A 3-D entire SRAM cell, based on a 0.35-microm current CMOS technology, is simulated in this work with a DEVICE simulator. The transient current, resulting from a heavy ion strike in the most sensitive region of the cell, is studied as a function of the LET value, the cell layout and the ion penetration depth. A definition of the critical charge is proposed and two new methods are presented to compute this basic amount of charge only using SPICE simulations. Numerical applications are performed with two different generations of submicron CMOS technologies, including the determination of the sensitive thicknesses

  1. In-chip fabrication of free-form 3D constructs for directed cell migration analysis

    Olsen, Mark Holm; Hjortø, Gertrud Malene; Hansen, Morten;

    2013-01-01

    Free-form constructs with three-dimensional (3D) microporosity were fabricated by two-photon polymerization inside the closed microchannel of an injection-molded, commercially available polymer chip for analysis of directed cell migration. Acrylate constructs were produced as woodpile topologies...... with a range of pore sizes from 5 × 5 μm to 15 × 15 μm and prefilled with fibrillar collagen. Dendritic cells seeded into the polymer chip in a concentration gradient of the chemoattractant CCL21 efficiently negotiated the microporous maze structure for pore sizes of 8 × 8 μm or larger. The cells...

  2. An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds

    Alexander Röder; Elena García-Gareta; Christina Theodoropoulos; Nikola Ristovski; Keith A. Blackwood; Woodruff, Maria A.

    2015-01-01

    The use of biopolymers as a three dimensional (3D) support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface ...

  3. Controlled Positioning of Cells in Biomaterials—Approaches Towards 3D Tissue Printing

    Sandra Hofmann

    2011-08-01

    Full Text Available Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extracellular matrices in a controlled manner is a process called bioprinting or biofabrication, which effectively uses principles of rapid prototyping combined with cell-loaded biomaterials, typically hydrogels. 3D tissue printing is an approach to manufacture functional tissue layer-by-layer that could be transplanted in vivo after production. This method is especially advantageous for stem cells since a controlled environment can be created to influence cell growth and differentiation. Using printed tissue for biotechnological and pharmacological needs like in vitro drug-testing may lead to a revolution in the pharmaceutical industry since animal models could be partially replaced by biofabricated tissues mimicking human physiology and pathology. This would not only be a major advancement concerning rising ethical issues but would also have a measureable impact on economical aspects in this industry of today, where animal studies are very labor-intensive and therefore costly. In this review, current controlled material and cell positioning techniques are introduced highlighting approaches towards 3D tissue printing.

  4. Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D

    MultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D. Cell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters. We describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage. Our data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models

  5. Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

    Chuang, Han-Ning; Lohaus, Raphaela; Hanisch, Uwe-Karsten; Binder, Claudia

    2013-01-01

    Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7. Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8. For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain

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

    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.

  7. Non-crimp 3D woven composites unit cell: from geometric modelling to damage simulation

    Bedogni, Enrico

    2013-01-01

    In the last twenty years, the research on composite materials has increased and many progresses have been made. However, there are still unresolved issues concerning the geometric modelling of a material at the meso-level (i.e. on a unit cell) and its damage simulation. In particular, the complexity of the internal geometry of some composite materials, such as 3D textiles, yields to new challenges for the research community. A correct definition of the internal structure in all the important ...

  8. Digital holography for recovering 3D shape of red blood cells

    Memmolo, P.; Miccio, L.; Merola, F.; Gennari, O.; Netti, P.; Ferraro, Pietro

    2015-07-01

    Full morphometric data analysis and 3D rendering of Red Blood Cells (RBCs) is provided by means of Digital Holography (DH) in combination with Optical Tweezers (OT). The proposed method is compared with a geometrical model of RBC in order to evaluate its accuracy and tested for many kinds of RBCs, from healthy ones with double-concavity to that with abnormal shapes. Applications in diagnostics are foreseen.

  9. Fish Stem Cell Cultures

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  10. Development of a 3D coculture system to study adipocyte and lymph node cell interactions

    Daya, S.; Loughlin, J.; MacQueen, H.

    2006-01-01

    We have developed a long term 3-dimensional coculture system with adipocytes and lymph node cells for the purpose of investigating interactions between these cells in vitro. Present experimental work with the culture system is aimed at introducing lymph node cells, in proportions similar to those found in intact lymph nodes, among differentiated adipocytes and observing interactions and the establishment of a spatial relationship between them. Co-cultures will be used to investigate the ly...

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

    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. Probabilistic Voxel-Fe model for single cell motility in 3D

    Borau, Carlos; Polacheck, William J; Kamm, Roger D; García-Aznar, José Manuel

    2015-01-01

    Background Cells respond to a variety of external stimuli regulated by the environment conditions. Mechanical, chemical and biological factors are of great interest and have been deeply studied. Furthermore, mathematical and computational models have been rapidly growing over the past few years, permitting researches to run complex scenarios saving time and resources. Usually these models focus on specific features of cell migration, making them only suitable to study restricted phenomena. Methods Here we present a versatile finite element (FE) cell-scale 3D migration model based on probabilities depending in turn on ECM mechanical properties, chemical, fluid and boundary conditions. Results With this approach we are able to capture important outcomes of cell migration such as: velocities, trajectories, cell shape and aspect ratio, cell stress or ECM displacements. Conclusions The modular form of the model will allow us to constantly update and redefine it as advancements are made in clarifying how cellular events take place. PMID:26290806

  13. Neural Stem Cells (NSCs in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI

    Alexandra Kourgiantaki

    2014-06-01

    Full Text Available Spinal cord injury, a traumatic disease characterised by a massive degeneration of neural tissue, was recently targeted for neuroregenerative interventions. Our approach is the development of pharmacologically pulsed neuroimplants using 3D collagen scaffolds hosting NSCs. We aim to monitor the properties of NSCs ex vivo and in vivo, using synthetic small molecules with neuroprotective and neurogenic properties. Synthetic, highly lipophilic CNS bioavailable small molecules, synthesized by our group (microneurotrophins, bind to neurotrophins receptors (Gravanis et al, Science Signaling, 2012, Calogeropoulou et al., J Med Chem., 2009. BNN27 can specifically interact with TrkA and p75NTR receptors activating specific signalling pathways controlling neuronal cell survival and neurogenesis (Charalampopoulos et al, PNAS, 2004, Lazaridis et al., PLoS Biol., 2011. We are seeding embryonic and adult mouse NSC on collagen 3D scaffolds of different composition (collagen, chondroitin-6-sulphate and gelatin and construction (size of pores and stiffness, testing cell behaviour (survival, proliferation or differentiation in basal conditions or pulsed with neurotrophins and/or microneurotrophins. Using the knock in sox2-egfp mice strain and fluorescence activated cell sorting (FACS analysis, we obtain NSCs cultures with a sox2-positive population more than 90% pure. We evaluate specific markers of proliferation (ki67 and/or differentiation (GFAP for glial cells, Tuj1 for mature neurons and O4 for oligodendrocytes: we are currently testing the possible effect of BNN27 on proliferation of cortical NSCs in 2D cultures (increased numbers of ki67 positive cells up to 12%. The composition and the structure of 3D scaffolds seem to play a significant functional role: scaffolds with a combined composition such as 50% collagen/50% gelatin and 92% collagen/8% chondroitin-6-sulphate support NSC survival since they sustain sox2 expression and propagate neurosphere formation

  14. 3D X-rays application for precision measurement of the cell structure of extruded polystyrene

    Lim, J. Y.; Kim, K. Y.; Shin, H. S.; Yeom, S.; Lee, S. E.

    2015-12-01

    While the thermal performance of existing insulation materials have been determined by blister gases, the thermal performance of future insulation materials will be dependent on the cell size and independent foam content as we use eco-friendly blister gases with a higher thermal conductivity. However, with the current technology we are only able to guess the whole cell size and independent foam content through SEM applied 2D fragmentary scanning but are still far from the level of accurate cell structure data extraction. Under this situation, we utilized X-ray CT scanned 3D images to identify and shape the cell structure and proposed a method of inferring the whole distribution and independent foam content as accurately as possible. According to X-ray CT scanning images and SEM images, the shape was similar but according to tracer applied CT scanning images, the cell size distribution was 380∼400 pm within the range of the general insulation diameter distribution which had the highest reliability. As for extrusion foaming polystyrene, we need additional image processing to identify the independent foam content as its density is too low. So, it is recommended to raise the 3D cell structure completeness of XPS by improving the scanning accuracy.

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

    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).

  16. Mechanical Properties of 3-D Printed Cellular Foams with triangular cells

    Bunga, Pratap Kumar

    In the present work, poly lactic acid (PLA) is used as a model system to investigate the mechanical behavior of 3-D printed foams with triangular cells. Solid PLA tension and compression specimens and foams made of PLA were fabricated using fused deposition 3-D printing technique. The solid PLA tension specimens were characterized for their densities and found to be about 10% lower in density as compared to their bulk counter parts. The triangular foams had a relative density of about 64%. The relationships between the structure of the foams and its deformation behavior under compression along two in-plane directions were characterized. Furthermore, simple finite element models were developed to understand the observed deformation behavior of triangular foams.

  17. 3D SURVEY AND AUGMENTED REALITY FOR CULTURAL HERITAGE. THE CASE STUDY OF AURELIAN WALL AT CASTRA PRAETORIA IN ROME

    M. Canciani

    2016-06-01

    Full Text Available The development of close-range photogrammetry has produced a lot of new possibility to study cultural heritage. 3D data acquired with conventional and low cost cameras can be used to document, investigate the full appearance, materials and conservation status, to help the restoration process and identify intervention priorities. At the same time, with 3D survey a lot of three-dimensional data are collected and analyzed by researchers, but there are a very few possibility of 3D output. The augmented reality is one of this possible output with a very low cost technology but a very interesting result. Using simple mobile technology (for iPad and Android Tablets and shareware software (in the case presented “Augment” it is possible to share and visualize a large number of 3D models with your own device. The case study presented is a part of an architecture graduate thesis, made in Rome at Department of Architecture of Roma Tre University. We have developed a photogrammetric survey to study the Aurelian Wall at Castra Praetoria in Rome. The surveys of 8000 square meters of surface have allowed to identify stratigraphy and construction phases of a complex portion of Aurelian Wall, specially about the Northern door of Castra. During this study, the data coming out of 3D survey (photogrammetric and topographic, are stored and used to create a reverse 3D model, or virtual reconstruction, of the Northern door of Castra. This virtual reconstruction shows the door in the Tiberian period, nowadays it's totally hidden by a curtain wall but, little and significative architectural details allow to know its original feature. The 3D model of the ancient walls has been mapped with the exact type of bricks and mortar, oriented and scaled according to the existing one to use augmented reality. Finally, two kind of application have been developed, one on site, were you can see superimposed the virtual reconstruction on the existing walls using the image

  18. A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion

    The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively. The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2) is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells. Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on tumor cell invasiveness

  19. A method for the evaluation of thousands of automated 3D stem cell segmentations.

    Bajcsy, P; Simon, M; Florczyk, S J; Simon, C G; Juba, D; Brady, M C

    2015-12-01

    There is no segmentation method that performs perfectly with any dataset in comparison to human segmentation. Evaluation procedures for segmentation algorithms become critical for their selection. The problems associated with segmentation performance evaluations and visual verification of segmentation results are exaggerated when dealing with thousands of three-dimensional (3D) image volumes because of the amount of computation and manual inputs needed. We address the problem of evaluating 3D segmentation performance when segmentation is applied to thousands of confocal microscopy images (z-stacks). Our approach is to incorporate experimental imaging and geometrical criteria, and map them into computationally efficient segmentation algorithms that can be applied to a very large number of z-stacks. This is an alternative approach to considering existing segmentation methods and evaluating most state-of-the-art algorithms. We designed a methodology for 3D segmentation performance characterization that consists of design, evaluation and verification steps. The characterization integrates manual inputs from projected surrogate 'ground truth' of statistically representative samples and from visual inspection into the evaluation. The novelty of the methodology lies in (1) designing candidate segmentation algorithms by mapping imaging and geometrical criteria into algorithmic steps, and constructing plausible segmentation algorithms with respect to the order of algorithmic steps and their parameters, (2) evaluating segmentation accuracy using samples drawn from probability distribution estimates of candidate segmentations and (3) minimizing human labour needed to create surrogate 'truth' by approximating z-stack segmentations with 2D contours from three orthogonal z-stack projections and by developing visual verification tools. We demonstrate the methodology by applying it to a dataset of 1253 mesenchymal stem cells. The cells reside on 10 different types of biomaterial

  20. Estimation of single cell volume from 3D confocal images using automatic data processing

    Chorvatova, A.; Cagalinec, M.; Mateasik, A.; Chorvat, D., Jr.

    2012-06-01

    Cardiac cells are highly structured with a non-uniform morphology. Although precise estimation of their volume is essential for correct evaluation of hypertrophic changes of the heart, simple and unified techniques that allow determination of the single cardiomyocyte volume with sufficient precision are still limited. Here, we describe a novel approach to assess the cell volume from confocal microscopy 3D images of living cardiac myocytes. We propose a fast procedure based on segementation using active deformable contours. This technique is independent on laser gain and/or pinhole settings and it is also applicable on images of cells stained with low fluorescence markers. Presented approach is a promising new tool to investigate changes in the cell volume during normal, as well as pathological growth, as we demonstrate in the case of cell enlargement during hypertension in rats.

  1. Many-faced cells and many-edged faces in 3D Poisson–Voronoi tessellations

    Motivated by recent new Monte Carlo data we investigate a heuristic asymptotic theory that applies to n-faced 3D Poisson–Voronoi cells in the limit of large n. We show how this theory may be extended to n-edged cell faces. It predicts the leading order large-n behavior of the average volume and surface area of the n-faced cell, and of the average area and perimeter of the n-edged face. Such a face is shown to be surrounded by a toroidal region of volume n/λ (with λ the seed density) that is void of seeds. Two neighboring cells sharing an n-edged face are found to have their seeds at a typical distance that scales as n−1/6 and whose probability law we determine. We present a new data set of 4 × 109 Monte Carlo generated 3D Poisson–Voronoi cells, larger than any before. Full compatibility is found between the Monte Carlo data and the theory. Deviations from the asymptotic predictions are explained in terms of subleading corrections whose powers in n we estimate from the data. (paper)

  2. Many-faced cells and many-edged faces in 3D Poisson-Voronoi tessellations

    Hilhorst, H. J.; Lazar, E. A.

    2014-10-01

    Motivated by recent new Monte Carlo data we investigate a heuristic asymptotic theory that applies to n-faced 3D Poisson-Voronoi cells in the limit of large n. We show how this theory may be extended to n-edged cell faces. It predicts the leading order large-n behavior of the average volume and surface area of the n-faced cell, and of the average area and perimeter of the n-edged face. Such a face is shown to be surrounded by a toroidal region of volume n/λ (with λ the seed density) that is void of seeds. Two neighboring cells sharing an n-edged face are found to have their seeds at a typical distance that scales as n-1/6 and whose probability law we determine. We present a new data set of 4 × 109 Monte Carlo generated 3D Poisson-Voronoi cells, larger than any before. Full compatibility is found between the Monte Carlo data and the theory. Deviations from the asymptotic predictions are explained in terms of subleading corrections whose powers in n we estimate from the data.

  3. 3D BREAST TISSUE CO-CULTURES FOR SCREENING MAMMARY CARCINOGENS - PHASE I

    Breast cancer is not a disease of individual cells, but principally a failure of cells and tissues to communicate properly. One communication mechanism that is frequently disrupted in breast cancer involves the hormone estrogen. Despite recognition that exposure to compound...

  4. Direct 3D-printing of cell-laden constructs in microfluidic architectures.

    Liu, Justin; Hwang, Henry H; Wang, Pengrui; Whang, Grace; Chen, Shaochen

    2016-04-21

    Microfluidic platforms have greatly benefited the biological and medical fields, however standard practices require a high cost of entry in terms of time and energy. The utilization of three-dimensional (3D) printing technologies has greatly enhanced the ability to iterate and build functional devices with unique functions. However, their inability to fabricate within microfluidic devices greatly increases the cost of producing several different devices to examine different scientific questions. In this work, a variable height micromixer (VHM) is fabricated using projection 3D-printing combined with soft lithography. Theoretical and flow experiments demonstrate that altering the local z-heights of VHM improved mixing at lower flow rates than simple geometries. Mixing of two fluids occurs as low as 320 μL min(-1) in VHM whereas the planar zigzag region requires a flow rate of 2.4 mL min(-1) before full mixing occurred. Following device printing, to further demonstrate the ability of this projection-based method, complex, user-defined cell-laden scaffolds are directly printed inside the VHM. The utilization of this unique ability to produce 3D tissue models within a microfluidic system could offer a unique platform for medical diagnostics and disease modeling. PMID:26980159

  5. Effects of electromagnetic field frequencies on chondrocytes in 3D cell-printed composite constructs.

    Yi, Hee-Gyeong; Kang, Kyung Shin; Hong, Jung Min; Jang, Jinah; Park, Moon Nyeo; Jeong, Young Hun; Cho, Dong-Woo

    2016-07-01

    In cartilage tissue engineering, electromagnetic field (EMF) therapy has been reported to have a modest effect on promoting cartilage regeneration. However, these studies were conducted using different frequencies of EMF to stimulate chondrocytes. Thus, it is necessary to investigate the effect of EMF frequency on cartilage formation. In addition to the stimulation, a scaffold is required to satisfy the characteristics of cartilage such as its hydrated and dense extracellular matrix, and a mechanical resilience to applied loads. Therefore, we 3D-printed a composite construct composed of a polymeric framework and a chondrocyte-laden hydrogel. Here, we observed frequency-dependent positive and negative effects on chondrogenesis using a 3D cell-printed cartilage tissue. We found that a frequency of 45 Hz promoted gene expression and secretion of extracellular matrix molecules of chondrocytes. In contrast, a frequency of 7.5 Hz suppressed chondrogenic differentiation in vitro. Additionally, the EMF-treated composite constructs prior to implantation showed consistent results with those of in vitro, suggesting that in vitro pre-treatment with different EMF frequencies provides different capabilities for the enhancement of cartilage formation in vivo. This correlation between EMF frequency and 3D-printed chondrocytes suggests the necessity for optimization of EMF parameters when this physical stimulus is applied to engineered cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1797-1804, 2016. PMID:26991030

  6. 3D cut-cell modelling for high-resolution atmospheric simulations

    Yamazaki, H; Nikiforakis, N

    2015-01-01

    With the recent, rapid development of computer technology, the resolution of atmospheric numerical models has increased substantially. As a result, steep gradients in mountainous terrain are now being resolved in high-resolution models. This results in large truncation errors in those models using terrain-following coordinates. In this study, a new 3D Cartesian coordinate non-hydrostatic atmospheric model is developed. A cut-cell representation of topography based on finite-volume discretization is combined with a cell-merging approach, in which small cut-cells are merged with neighboring cells either vertically or horizontally. In addition, a block-structured mesh-refinement technique achieves a variable resolution on the model grid with the finest resolution occurring close to the terrain surface. The model successfully reproduces a flow over a 3D bell-shaped hill that shows a good agreement with the flow predicted by the linear theory. The ability of the model to simulate flows over steep terrain is demons...

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

    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.

  8. Using LIDO to handle 3D cultural heritage documentation data provenance

    Pitzalis, Denis; Niccolucci, Franco; Cord, Matthieu

    2011-01-01

    International audience It is important for Digital Libraries (DL) to be flexible in exposing their content. Typically a DL provides a search/browse interface which allows resources to be found and a service to make the data available for harvesting from/to other DLs. This kind of communication is possible because the structures of different DLs are expressed following formal specifications. In particular in Cultural Heritage, where we need to describe an extremely heterogeneous environment...

  9. The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments

    Wrzesinski, Krzysztof; Rogowska-Wrzesinska, Adelina; Borkowski, Kamil;

    2014-01-01

    cholesterol and urea synthesis are increased underpinning the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in G0. Transport around the cell – along the microtubules, through the nuclear pore and in various types of...

  10. Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells

    Gruene, M; Deiwick, A; Koch, L; Schlie, S; Unger, C; Chichkov, B N [Nanotechnology Department, Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover (Germany); Pflaum, M; Wilhelmi, M; Haverich, A, E-mail: m.gruene@lzh.de [Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover (Germany)

    2011-03-15

    Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

  11. Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells

    Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

  12. Isotropic 3D Nuclear Morphometry of Normal, Fibrocystic and Malignant Breast Epithelial Cells Reveals New Structural Alterations

    Nandakumar, Vivek; Kelbauskas, Laimonas; Hernandez, Kathryn F.; Lintecum, Kelly M.; Senechal, Patti; Bussey, Kimberly J.; Davies, Paul C.W.; Johnson, Roger H.; Meldrum, Deirdre R.

    2012-01-01

    Background Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading ...

  13. Generating 3D tissue constructs with mesenchymal stem cells and a cancellous bone graft for orthopaedic applications

    Arca, Turkan; Genever, Paul [Department of Biology, University of York, York, YO10 5DD (United Kingdom); Proffitt, Joanne, E-mail: paul.genever@york.ac.uk [TSL Centre of Biologics, Covidien, Allerton Bywater, Castleford, WF10 2DB (United Kingdom)

    2011-04-15

    Bone matrix (BM) is an acellular crosslinked porcine-derived cancellous bone graft, and therefore may provide advantages over other synthetic and naturally derived materials for use in orthopaedic surgery. Here, we analysed the potential of BM to support the growth and differentiation of primary human multipotent stromal cells/mesenchymal stem cells (MSCs) in order to predict in vivo bone regeneration events. Imaging with laser scanning confocal microscopy and scanning electron microscopy showed that 1 day after static seeding, a dense population of viable MSCs could be achieved on scaffolds suggesting they could be used for in vivo delivery of cells to the implant site. Long-term growth analysis by confocal imaging and histology demonstrated that BM was permissive to the growth and the 3D population of primary MSCs and an enhanced green fluorescent protein expressing osteosarcoma cell line, eGFP.MG63s, over several days in culture. Measurement of alkaline phosphatase (ALP) activities and mRNA expression levels of osteogenic markers (Runx-2, ALP, collagen type I, osteonectin, osteocalcin and osteopontin) indicated that BM supported osteogenesis of MSCs when supplemented with osteogenic stimulants. Upregulation of some of these osteogenic markers on BM, but not on tissue culture plastic, under non-osteogenic conditions suggested that BM also had osteoinductive capacities.

  14. Enabling Flexible Polymer Tandem Solar Cells by 3D Ptychographic Imaging

    Dam, Henrik Friis; Andersen, Thomas Rieks; Pedersen, Emil Bøje Lind;

    2015-01-01

    one after the other by wet processing leaves plenty of room for error and the process development calls for an analytical technique that enables 3D reconstruction of the layer stack with the possibility to probe thickness, density, and chemistry of the individual layers in the stack. The use of......The realization of a complete tandem polymer solar cell under ambient conditions using only printing and coating methods on a flexible substrate results in a fully scalable process but also requires accurate control during layer formation to succeed. The serial process where the layers are added...

  15. 3-D slug flow heat transfer analysis of coupled coolant cells in finite LMFBR bundles

    A three-dimensional single region slug flow heat transfer analysis for finite LMFBR rod bundles using a classical analytical solution method has been performed. According to the isolated single cell analysis, the results show that the peripheral clad temperature variation as well as the thermal entrance length are strongly dependent upon the degree of irregularity displayed by various coolant geometries. Since under the present LMFBR conditions, fully-developed temperature fields may hardly be established in such characteristic rod bundle regions, a 3-D heat transfer analysis seems to be mandatory. This implies that the results of fully developed heat transfer analyses are by far too conservative

  16. A harmonic polynomial cell (HPC) method for 3D Laplace equation with application in marine hydrodynamics

    We propose a new efficient and accurate numerical method based on harmonic polynomials to solve boundary value problems governed by 3D Laplace equation. The computational domain is discretized by overlapping cells. Within each cell, the velocity potential is represented by the linear superposition of a complete set of harmonic polynomials, which are the elementary solutions of Laplace equation. By its definition, the method is named as Harmonic Polynomial Cell (HPC) method. The characteristics of the accuracy and efficiency of the HPC method are demonstrated by studying analytical cases. Comparisons will be made with some other existing boundary element based methods, e.g. Quadratic Boundary Element Method (QBEM) and the Fast Multipole Accelerated QBEM (FMA-QBEM) and a fourth order Finite Difference Method (FDM). To demonstrate the applications of the method, it is applied to some studies relevant for marine hydrodynamics. Sloshing in 3D rectangular tanks, a fully-nonlinear numerical wave tank, fully-nonlinear wave focusing on a semi-circular shoal, and the nonlinear wave diffraction of a bottom-mounted cylinder in regular waves are studied. The comparisons with the experimental results and other numerical results are all in satisfactory agreement, indicating that the present HPC method is a promising method in solving potential-flow problems. The underlying procedure of the HPC method could also be useful in other fields than marine hydrodynamics involved with solving Laplace equation

  17. In-cell maintenance by manipulator arm with 3D workspace information recreated by laser rangefinder

    Highlights: → We developed a remote control system for maintenance of in-cell type fuel fabrication equipment. → The system display recreated three-dimensional information of the workspace from data obtained by laser rangefinder and conventional cameras. It has allowed us to operate a manipulator arm remotely with several control modes. → We implemented remote handling experiments using mock up equipment. Performance was compared for remote operation conducted using several different display and operation modes. → It was observed that integration of 3D information from the laser rangefinder reduced operation time and reinforced visual information during remote operation. - Abstract: We developed a remote control system for maintenance of in-cell type fuel fabrication equipment. The system display recreated three-dimensional information of the workspace from data obtained by laser rangefinder and conventional cameras. It has allowed us to operate a manipulator arm remotely with several control modes. In order to evaluate the effectiveness and usefulness of developed system, we implemented remote handling experiments using mock up equipment. Performance was compared for remote operation conducted using several different display and operation modes. We confirmed that the system is able to maintain in-cell fuel fabrication equipment in each display and operation mode. Times required to complete the remote operations were collected and compared in each mode. It was observed that integration of 3D information from the laser rangefinder reduced operation time and reinforced visual information during remote operation.

  18. A harmonic polynomial cell (HPC) method for 3D Laplace equation with application in marine hydrodynamics

    Shao, Yan-Lin, E-mail: yanlin.shao@dnvgl.com; Faltinsen, Odd M.

    2014-10-01

    We propose a new efficient and accurate numerical method based on harmonic polynomials to solve boundary value problems governed by 3D Laplace equation. The computational domain is discretized by overlapping cells. Within each cell, the velocity potential is represented by the linear superposition of a complete set of harmonic polynomials, which are the elementary solutions of Laplace equation. By its definition, the method is named as Harmonic Polynomial Cell (HPC) method. The characteristics of the accuracy and efficiency of the HPC method are demonstrated by studying analytical cases. Comparisons will be made with some other existing boundary element based methods, e.g. Quadratic Boundary Element Method (QBEM) and the Fast Multipole Accelerated QBEM (FMA-QBEM) and a fourth order Finite Difference Method (FDM). To demonstrate the applications of the method, it is applied to some studies relevant for marine hydrodynamics. Sloshing in 3D rectangular tanks, a fully-nonlinear numerical wave tank, fully-nonlinear wave focusing on a semi-circular shoal, and the nonlinear wave diffraction of a bottom-mounted cylinder in regular waves are studied. The comparisons with the experimental results and other numerical results are all in satisfactory agreement, indicating that the present HPC method is a promising method in solving potential-flow problems. The underlying procedure of the HPC method could also be useful in other fields than marine hydrodynamics involved with solving Laplace equation.

  19. Optimizing stem cell culture.

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-11-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane. PMID:20803548

  20. Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy.

    Chang, Lingqian; Gallego-Perez, Daniel; Zhao, Xi; Bertani, Paul; Yang, Zhaogang; Chiang, Chi-Ling; Malkoc, Veysi; Shi, Junfeng; Sen, Chandan K; Odonnell, Lynn; Yu, Jianhua; Lu, Wu; Lee, L James

    2015-08-01

    Current transfection technologies lead to significant inter-clonal variations. Previously we introduced a unique electrotransfection technology, Nanochannel-Electroporation (NEP), which can precisely and benignly transfect small cell populations (~100-200 cells) with single-cell resolution. Here we report on the development of a novel 3D NEP system for large scale transfection. A properly-engineered array of nanochannels, capable of handling/transfecting ~60 000 cells cm(-2), was fabricated using cleanroom technologies. Positive dielectrophoresis was used to selectively position cells on the nanochannels, thus allowing highly efficient transfection. Single-cell dosage control was demonstrated using both small and large molecules, and different cell types. The potential clinical relevance of this system was tested with difficult-to-transfect natural killer cell suspensions, and plasmids encoding for the chimeric antigen receptor (CAR), a model of high relevance for adoptive immunotherapy. Our results show significantly higher CAR transfection efficiencies for the DEP-NEP system (>70% vs. <30%), as well as enhanced cell viabilities. PMID:26105628

  1. Three-Dimensional Cell Culture: A Breakthrough in Vivo

    Delphine Antoni

    2015-03-01

    Full Text Available Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design.

  2. Prompt gamma-ray 3D-imaging for cultural heritage purposes

    Schulze, Ralf

    2010-07-01

    The development of new, and the enhancement of existing element-sensitive imaging methods utilizing neutrons of different energy regions was the aim of the European ANCIENT CHARM project. During the present work the setup for Prompt Gamma-ray Activation Analysis (PGAA) at the research reactor FRM2 in Garching near Munich was modified to enable the spatial mapping of elemental abundances in the analysed samples. Because the PGAA setup at FRM2 was under construction at the beginning of the project first tests and the development of calibration and measurement procedures for the new imaging method were done by the PGAA group at the Budapest Research Reactor in cooperation with the Institute for Nuclear Physics of the University of Cologne. Due to the higher neutron flux at the PGAA setup at FRM2 the equipment was transferred from the Budapest Research Reactor to FRM2 after the PGAA setup at FRM2 started its regular operation. After further optimizations and the characterization of the setup, measurements were started on replicas of real archaeological objects before several measurements on real objects were performed and analysed. Several measurement configurations were applied. Additional to 2D and 3D imaging measurements a new application for the measurement of thin surface layers in the order of a few 100 {mu}m was developed. For the quantitative analysis of elemental distributions the exact knowledge of the neutron flux at each measured position in the analysed sample has to be known. Based on the well-established cold Neutron Tomography (NT) method a method and software have been developed, which enables the calculation of the neutron flux inside samples with the map of attenuation properties obtained through NT. A new data acquisition system was developed for the regular operation of the PGAA setup at FRM2, which supports traditional bulk PGAA measurements as well as measurements in the new imaging configuration. The high automation of the system allows a

  3. Prompt gamma-ray 3D-imaging for cultural heritage purposes

    The development of new, and the enhancement of existing element-sensitive imaging methods utilizing neutrons of different energy regions was the aim of the European ANCIENT CHARM project. During the present work the setup for Prompt Gamma-ray Activation Analysis (PGAA) at the research reactor FRM2 in Garching near Munich was modified to enable the spatial mapping of elemental abundances in the analysed samples. Because the PGAA setup at FRM2 was under construction at the beginning of the project first tests and the development of calibration and measurement procedures for the new imaging method were done by the PGAA group at the Budapest Research Reactor in cooperation with the Institute for Nuclear Physics of the University of Cologne. Due to the higher neutron flux at the PGAA setup at FRM2 the equipment was transferred from the Budapest Research Reactor to FRM2 after the PGAA setup at FRM2 started its regular operation. After further optimizations and the characterization of the setup, measurements were started on replicas of real archaeological objects before several measurements on real objects were performed and analysed. Several measurement configurations were applied. Additional to 2D and 3D imaging measurements a new application for the measurement of thin surface layers in the order of a few 100 μm was developed. For the quantitative analysis of elemental distributions the exact knowledge of the neutron flux at each measured position in the analysed sample has to be known. Based on the well-established cold Neutron Tomography (NT) method a method and software have been developed, which enables the calculation of the neutron flux inside samples with the map of attenuation properties obtained through NT. A new data acquisition system was developed for the regular operation of the PGAA setup at FRM2, which supports traditional bulk PGAA measurements as well as measurements in the new imaging configuration. The high automation of the system allows a

  4. Cell Culture Made Easy.

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  5. 3DMADMAC|SPECTRAL: Hardware and Software Solution for Integrated Digitization of 3D Shape, Multispectral Color and BRDF for Cultural Heritage Documentation

    Robert Sitnik

    2011-12-01

    Full Text Available In this article a new 3D measurement system along with the study on 3D printing technology is presented from the perspective of quality of reproduction. In the first part of the paper the 3DMADMAC|SPECTRAL system which integrates 3D shape with additional color and angular reflectance measurement capabilities is presented (see Figure 1. The shape measurement system is based on structured light projection with the use of a DLP projector. The 3D shape measurement method is based on sinusoidal fringes and Gray codes projection. Color is being measured using multispectral images with a set of interference filters to separate spectral channels. Additionally the set up includes an array of compact light sources for measuring angular reflectance based on image analysis and 3D data processing. All three components of the integrated system use the same greyscale camera as a detector. The purpose of the system is to obtain complete information about shape, color and reflectance characteristic of mea sured surface, especially for cultural heritage objects - in order to create high quality 3D documentation. In the second part of the paper the 3D printing technology will be tested on real measured cultural heritage objects. Tests allow to assess measurement and color accuracy of reproduction by selected 3D printing technology and shed some light on how current 3D printing technology can be applied into cultural heritage.

  6. Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.

    Reid, John A; Mollica, Peter A; Johnson, Garett D; Ogle, Roy C; Bruno, Robert D; Sachs, Patrick C

    2016-01-01

    The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive 'off-the-shelf' commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, 'luer-lock' syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an

  7. A 3D model of ovarian cancer cell lines on peptide nanofiber scaffold to explore the cell–scaffold interaction and chemotherapeutic resistance of anticancer drugs

    Zehong Yang

    2011-02-01

    Full Text Available Zehong Yang1, Xiaojun Zhao1,21Nanomedicine Laboratory, West China Hospital and Institute for Nanobiomedical Technology and Membrane Biology, Sichuan University, Chengdu, People’s Republic of China; 2Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USAAbstract: RADA16-I peptide hydrogel, a type of nanofiber scaffold derived from self-assembling peptide RADA16-I, has been extensively applied to regenerative medicine and tissue repair in order to develop novel nanomedicine systems. In this study, using RADA16-I peptide hydrogel, a three-dimensional (3D cell culture model was fabricated for in vitro culture of three ovarian cancer cell lines. Firstly, the peptide nanofiber scaffold was evaluated by transmission electron microscopy and atom force microscopy. Using phase contrast microscopy, the appearance of the representative ovarian cancer cells encapsulated in RADA16-I peptide hydrogel on days 1, 3, and 7 in 24-well Petri dishes was illustrated. The cancer cell–nanofiber scaffold construct was cultured for 5 days, and the ovarian cancer cells had actively proliferative potential. The precultured ovarian cancer cells exhibited nearly similar adhesion properties and invasion potentials in vitro between RADA16-I peptide nanofiber and type I collagen, which suggested that RADA16-I peptide hydrogel had some similar characteristics to type I collagen. The precultured ovarian cancer cells had two-fold to five-fold higher anticancer drug resistance than the conventional two-dimensional Petri dish culture. So the 3D cell model on peptide nanofiber scaffold is an optimal type of cell pattern for anticancer drug screening and tumor biology.Keywords: 3D culture, anticancer drug, nanofiber scaffold, cell viability, ovarian cancer

  8. One-step fabrication of 3D silver paste electrodes into microfluidic devices for enhanced droplet-based cell sorting

    Lang Rao

    2015-05-01

    Full Text Available 3D microelectrodes are one-step fabricated into a microfluidic droplet separator by filling conductive silver paste into PDMS microchambers. The advantages of 3D silver paste electrodes in promoting droplet sorting accuracy are systematically demonstrated by theoretical calculation, numerical simulation and experimental validation. The employment of 3D electrodes also helps to decrease the droplet sorting voltage, guaranteeing that cells encapsulated in droplets undergo chip-based sorting processes are at better metabolic status for further potential cellular assays. At last, target droplet containing single cell are selectively sorted out from others by an appropriate electric pulse. This method provides a simple and inexpensive alternative to fabricate 3D electrodes, and it is expected our 3D electrode-integrated microfluidic droplet separator platform can be widely used in single cell operation and analysis.

  9. Has 3-D conformal radiotherapy (3D CRT) improved the local tumour control for stage I non-small cell lung cancer?

    Aims and background: The high local failure rates observed after radiotherapy in stage I non-small cell lung cancer (NSCLC) may be improved by the use of 3-dimensional conformal radiotherapy (3D CRT). Materials and methods: The case-records of 113 patients who were treated with curative 3D CRT between 1991 and 1999 were analysed. No elective nodal irradiation was performed, and doses of 60 Gy or more, in once-daily fractions of between 2 and 3 Gy, were prescribed. Results: The median actuarial survival of patients was 20 months, with 1-, 3- and 5-year survival of 71, 25 and 12%, respectively. Local disease progression was the cause of death in 30% of patients, and 22% patients died from distant metastases. Grade 2-3 acute radiation pneumonitis (SWOG) was observed in 6.2% of patients. The median actuarial local progression-free survival (LPFS) was 27 months, with 85 and 43% of patients free from local progression at 1 and 3 years, respectively. Endobronchial tumour extension significantly influenced LPFS, both on univariate (P=0.023) and multivariate analysis (P=0.023). The median actuarial cause-specific survival (CSS) was 19 months, and the respective 1- and 3-year rates were 72 and 30%. Multivariate analysis showed T2 classification (P=0.017) and the presence of endobronchial tumour extension (P=0.029) to be adverse prognostic factors for CSS. On multivariate analysis, T-stage significantly correlated with distant failure (P=0.005). Conclusions: Local failure rates remain substantial despite the use of 3D CRT for stage I NSCLC. Additional improvements in local control can come about with the use of radiation dose escalation and approaches to address the problem of tumour mobility

  10. Adaptive geometric tessellation for 3D reconstruction of anisotropically developing cells in multilayer tissues from sparse volumetric microscopy images.

    Anirban Chakraborty

    Full Text Available The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2-4 slices/cell and wide range of cell shapes and sizes. The proposed method, named as the 'Adaptive Quadratic Voronoi Tessellation' (AQVT, is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells.

  11. Responsiveness to PI3K and MEK inhibitors in breast cancer. Use of a 3D culture system to study pathways related to hormone independence in mice.

    Maria Laura Polo

    Full Text Available 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

  12. Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity.

    Blaeser, Andreas; Duarte Campos, Daniela Filipa; Puster, Uta; Richtering, Walter; Stevens, Molly M; Fischer, Horst

    2016-02-01

    A microvalve-based bioprinting system for the manufacturing of high-resolution, multimaterial 3D-structures is reported. Applying a straightforward fluid-dynamics model, the shear stress at the nozzle site can precisely be controlled. Using this system, a broad study on how cell viability and proliferation potential are affected by different levels of shear stress is conducted. Complex, multimaterial 3D structures are printed with high resolution. This work pioneers the investigation of shear stress-induced cell damage in 3D bioprinting and might help to comprehend and improve the outcome of cell-printing studies in the future. PMID:26626828

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

    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

  14. Temperature distributions in the laser-heated diamond anvil cell from 3-D numerical modeling

    We present TempDAC, a 3-D numerical model for calculating the steady-state temperature distribution for continuous wave laser-heated experiments in the diamond anvil cell. TempDAC solves the steady heat conduction equation in three dimensions over the sample chamber, gasket, and diamond anvils and includes material-, temperature-, and direction-dependent thermal conductivity, while allowing for flexible sample geometries, laser beam intensity profile, and laser absorption properties. The model has been validated against an axisymmetric analytic solution for the temperature distribution within a laser-heated sample. Example calculations illustrate the importance of considering heat flow in three dimensions for the laser-heated diamond anvil cell. In particular, we show that a “flat top” input laser beam profile does not lead to a more uniform temperature distribution or flatter temperature gradients than a wide Gaussian laser beam

  15. Dynamic 3D cell rearrangements guided by a fibronectin matrix underlie somitogenesis.

    Gabriel G Martins

    Full Text Available Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

  16. Effectiveness of octenidine and chlorhexidine in the artificially contaminated 3-D-culture of human keratinocytes

    Kramer, Axel

    2009-12-01

    Full Text Available Epidermis-Equivalents (EpiDerm derived from human keratinocytes were obtained after 14 d air-lift-culture. These In-vitro-models were used for the demonstration of a possible postantiseptic effect against the test microorganisms Pseudomonas aeruginosa and Staphylococcus aureus after 5 min pre-incubation of the surface of the epidermis with equimolar concentrations of the antiseptic agents chlorhexidine digluconate (CHX and octenidine dihydrochloride (OCT of 1.6 mmol/l. Both antiseptics adhere to the surface of EpiDerm. Each active agent cannot be removed by washing with phosphate-buffered saline (PBS and remains microbiocidal active. EpiDerm-OCT was more effective after 30 min at room temperature against 106 cfu/ml Staphylococcus aureus resulting 3.2–4 log10 reduction, but not for EpiDerm-CHX producing only 2–2.4 log10 reduction. A maximum of 0.6–1.2 log10 reduction was found against Pseudomonas aeruginosa after 30 min exposure in EpiDerm-OCT, but there was no or a neglectable microbiocidal activity of 0–0.2 log10 reduction using EpiDerm-CHX. The postantiseptic effect of OCT is superior to EpiDerm-bound CHX comparing equimolar concentrations of active agent used for pre-incubation of EpiDerm. Treatment of EpiDerm with OCT alone or in combination with test organisms resulted in no cytotoxic effect in viable keratinocytes. In contrast to that the test organism Staphylococcus aureus, the active agent CHX alone or in combination with both test organisms demonstrated a cytotoxic activity reducing the viability of basal keratinocytes more than 10%. Therefore, OCT is more tolerated than CHX by basal keratinocytes of the presented In-vitro-model EpiDerm.

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

    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.

  18. Complete factorial design experiment for 3D load cell instrumented crank validation.

    Omar, Valle-Casas; Rafael, Dalazen; Vinicius, Cene; Alexandre, Balbinot

    2015-08-01

    Developing of instrumentation systems for sport medicine is a promising area, that's why this research evaluates the design of a new instrumented crank arm prototype for a race bicycle projecting an experiment for indoor - outdoor comparison. This study investigated the viability of an instrumentation 3D load cell for force measurement crank, implementing a design of experiment. A Complete factorial design experiment was developed for data validation, with an Analysis of Variance (ANOVA) throwing significant results for controlled factors with response variables rms, mean and variance. A software routine allowed to obtained system variables metrics for Symmetry and Cadence analysis, which came out from Effective force bilateral comparing and speed computation. Characterization allowed achieving calibration curves that were used for data conversion in force projection channels with a linearity error of 0.29% (perpendicular), 0.55% (parallel) and 0.10% (lateral). Interactions of factors resulted significant mainly for indoor tests in symmetry and cadence was significant in interactions generally for outdoor tests. Implemented system was able to generate Effective Force graph for 3D plot symmetry analysis, torque and power symmetry for specialist's analysis. PMID:26737085

  19. A MULTISCALE APPROACH TO THE REPRESENTATION OF 3D IMAGES, WITH APPLICATION TO POLYMER SOLAR CELLS

    Ralf Thiedmann

    2011-03-01

    Full Text Available A multiscale approach to the description of geometrically complex 3D image data is proposed which distinguishes between morphological features on a ‘macro-scale’ and a ‘micro-scale’. Since our method is mainly tailored to nanostructures observed in composite materials consisting of two different phases, an appropriate binarization of grayscale images is required first. Then, a morphological smoothing is applied to extract the structural information from binarized image data on the ‘macro-scale’. A stochastic algorithm is developed for the morphologically smoothed images whose goal is to find a suitable representation of the macro-scale structure by unions of overlapping spheres. Such representations can be interpreted as marked point patterns. They lead to an enormous reduction of data and allow the application of well-known tools from point-process theory for their analysis and structural modeling. All those voxels which have been ‘misspecified’ by the morphological smoothing and subsequent representation by unions of overlapping spheres are interpreted as ‘micro-scale’ structure. The exemplary data sets considered in this paper are 3D grayscale images of photoactive layers in hybrid solar cells gained by electron tomography. These composite materials consist of two phases: a polymer phase and a zinc oxide phase. The macro-scale structure of the latter is represented by unions of overlapping spheres.

  20. A Parallelized 3D Particle-In-Cell Method With Magnetostatic Field Solver And Its Applications

    Hsu, Kuo-Hsien; Chen, Yen-Sen; Wu, Men-Zan Bill; Wu, Jong-Shinn

    2008-10-01

    A parallelized 3D self-consistent electrostatic particle-in-cell finite element (PIC-FEM) code using an unstructured tetrahedral mesh was developed. For simulating some applications with external permanent magnet set, the distribution of the magnetostatic field usually also need to be considered and determined accurately. In this paper, we will firstly present the development of a 3D magnetostatic field solver with an unstructured mesh for the flexibility of modeling objects with complex geometry. The vector Poisson equation for magnetostatic field is formulated using the Galerkin nodal finite element method and the resulting matrix is solved by parallel conjugate gradient method. A parallel adaptive mesh refinement module is coupled to this solver for better resolution. Completed solver is then verified by simulating a permanent magnet array with results comparable to previous experimental observations and simulations. By taking the advantage of the same unstructured grid format of this solver, the developed PIC-FEM code could directly and easily read the magnetostatic field for particle simulation. In the upcoming conference, magnetron is simulated and presented for demonstrating the capability of this code.

  1. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    Chih-Hao Chang

    Full Text Available Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP techniques. A self-developed 3D printer with laser-aided gelling (LAG process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w. Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

  2. 3-D-conformal radiation therapy for pediatric giant cell tumors of the skull base

    Hug, E.B. [Massachusetts General Hospital, Boston, MA (United States). Dept. of Radiation Oncology; Harvard Univ., Cambridge, MA (United States). Cyclotron Lab.; Dartmouth Hitchcock Medical Center, Lebanon, NH (United States). Section of Radiation Oncology; Muenter, M.W.; Vries, A. de [Massachusetts General Hospital, Boston, MA (United States). Dept. of Radiation Oncology; Adams, J.A.; Munzenrider, J.E. [Massachusetts General Hospital, Boston, MA (United States). Dept. of Radiation Oncology; Harvard Univ., Cambridge, MA (United States). Cyclotron Lab.; Rosenberg, A.E. [Massachusetts General Hospital, Boston, MA (United States). Dept. of Pathology

    2002-05-01

    Background: Giant cell tumors (GCT) of the base of skull are rare neoplasms. This report reviews the treatment of four pediatric patients presenting with aggressive giant cell tumor, using fractionated and combined, conformal proton and photon radiation therapy at Massachusetts General Hospital and Harvard Cyclotron Laboratory. Patients and Methods: Three female patients and one adolescent male, ages 10-15 years, had undergone prior, extensive surgical resection(s) and were treated for either primary (two patients) or recurrent (two patients) disease. Gross residual tumor was evident in three patients and microscopic disease suspected in one patient. Combined proton and photon radiation theory was based on three-dimensional (3-D) planning, consisting of fractionated treatment, one fraction per day at 1.8 CGE (cobalt-gray equivalent) to total target doses of 57.6, 57.6, 59.4, and 61.2 Gy/CGE. Results: With observation times of 3.1 years, 3.3, 5.3, and 5.8 years, all four patients were alive and well and remained locally controlled without evidence of recurrent disease. Except for one patient with partial pituitary insufficiency following radiotherapy for sellar recurrent disease, thus far no late effects attributable to radiation therapy have been observed. Conclusions: 3-D conformal radiation therapy offers a realistic chance of tumor control for aggressive giant cell tumor in the skull base, either postoperatively or at time of recurrence. Conformal treatment techniques allow the safe delivery of relatively high radiation doses in the pediatric patient without apparent increase of side effects. (orig.)

  3. Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model.

    Stine Krog Frandsen

    Full Text Available Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy-and normal cell sensitivity.Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29, a bladder transitional cell carcinoma (SW780, and a breast adenocarcinoma (MDA-MB231, as well as on primary normal human dermal fibroblasts (HDF-n.The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p<0.0001 or electrochemotherapy using bleomycin (p<0.0001. Strikingly, the size of normal fibroblast spheroids was neither affected after calcium electroporation nor electrochemotherapy using bleomycin, indicating that calcium electroporation, like electrochemotherapy, will have limited adverse effects on the surrounding normal tissue when treating with calcium electroporation. The intracellular ATP level, which has previously been shown to be depleted after calcium electroporation, was measured in the spheroids after treatment. The results showed a dramatic decrease in the intracellular ATP level (p<0.01 in all four spheroid types-malignant as well as normal.In conclusion, calcium electroporation seems to be more effective in inducing cell death in cancer cell spheroids than in a normal fibroblast spheroid, even though intracellular ATP level is depleted in all spheroid types after treatment. These results may indicate an important therapeutic window for this therapy; although further studies are needed in vivo and in patients to investigate the effect of calcium electroporation on surrounding normal tissue when

  4. Isotropic 3D nuclear morphometry of normal, fibrocystic and malignant breast epithelial cells reveals new structural alterations.

    Vivek Nandakumar

    Full Text Available BACKGROUND: Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria. METHODOLOGY: We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure. PRINCIPAL FINDINGS: We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations. CONCLUSIONS: Our results provide a new perspective on nuclear

  5. Atypical pyramidal cells in epileptic human cortex: CFLS and 3-D reconstructions.

    Belichencko, P; Dahlström, A; von Essen, C; Lindström, S; Nordborg, C; Sourander, P

    1992-09-01

    Epileptic temporal cortices, removed from 3 patients with intractable partial epilepsy (IPE) during neurosurgery, were studied. Pyramidal neurons (40-50 per slice) in laminae III, V and white matter, were injected with lucifer yellow. Samples were examined in a confocal laser scanning microscope (Biorad 600) and individual cells scanned at 0.1-1 microns incremental levels. 2-D maximal linear projection was used for overview. Frames (50-60) of scanned neurons were transformed into 3-D volumes, using VoxelView software on a Silicone Graphics workstation and rotated. All samples contained neurons with duplicated apical dendrites, additional basal dendrites or were misplaced in a horizontal position in the white matter. The relation between these preliminary observations and the disease is discussed. PMID:1421134

  6. Parallel 3D Finite Element Particle-in-Cell Simulations with Pic3P

    Candel, A.; Kabel, A.; Lee, L.; Li, Z.; Ng, C.; Schussman, G.; Ko, K.; /SLAC; Ben-Zvi, I.; Kewisch, J.; /Brookhaven

    2009-06-19

    SLAC's Advanced Computations Department (ACD) has developed the parallel 3D Finite Element electromagnetic Particle-In-Cell code Pic3P. Designed for simulations of beam-cavity interactions dominated by space charge effects, Pic3P solves the complete set of Maxwell-Lorentz equations self-consistently and includes space-charge, retardation and boundary effects from first principles. Higher-order Finite Element methods with adaptive refinement on conformal unstructured meshes lead to highly efficient use of computational resources. Massively parallel processing with dynamic load balancing enables large-scale modeling of photoinjectors with unprecedented accuracy, aiding the design and operation of next-generation accelerator facilities. Applications include the LCLS RF gun and the BNL polarized SRF gun.

  7. Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers

    Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs. (paper)

  8. Novel carbocyclic curcumin analog CUR3d modulates genes involved in multiple apoptosis pathways in human hepatocellular carcinoma cells.

    Bhullar, Khushwant S; Jha, Amitabh; Rupasinghe, H P Vasantha

    2015-12-01

    Anticancer activity of a novel curcumin analog (E)-2-(4-hydroxy-3-methoxybenzylidene)-5-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)cyclopentanone (CUR3d) was studied using a human hepatocellular carcinoma cell line (HepG2). The results showed that CUR3d completely inhibits the tumor cell proliferation in a dose- and time-dependent manner. CUR3d at 100 μmol/L activated the pro-apoptotic caspase-3 along with downregulation of anti-apoptotic BIRC5 and Bcl2. CUR3d treatment controlled the cancer cell growth by downregulating the expression of PI3K/Akt (Akt1, Akt2) pathway along with NF-κB. CUR3d down-regulated the members of epidermal growth receptor family (EGFR, ERBB3, ERBB2) and insulin like growth receptors (IGF1, IGF-1R, IGF2). This correlated with the downregulation of G-protein (RHOA, RHOB) and RAS (ATF2, HRAS, KRAS, NRAS) pathway signaling. CUR3d also arrested cell cycle via inhibition of CDK2, CDK4, CDK5, CDK9, MDM2, MDM4 and TERT genes. Cell cycle essential aurora kinases (AURKα, AURKβ) and polo-like kinases (PLK1, PLK2, PLK3) were also modulated by CUR3d. Topoisomerases (TOP2α, TOP2β), important factors in cancer cell immortality, as well as HIF-1α were downregulated following CUR3d treatment. The expression of protein kinase-C family (PRKC-A, PRKC-D, PRKC-E) was also attenuated by CUR3d. The downregulation of histone deacetylases (Class I, II, IV) and PARP I further strengthened the anticancer efficacy of CUR3d. Downregulation of carcinogenic cathepsins (CTSB, CTSD) and heat shock proteins exhibited CUR3d's potency as a potential immunological adjuvant. Finally, the non-toxic manifestation of CUR3d in healthy liver and lung cells along with downregulation of drug resistant gene ABCC1 further warrant need for advance investigations. PMID:26409325

  9. Using Polymer Confinement for Stem Cell Differentiation: 3D Printed vs Molded Scaffolds

    Rafailovich, Miriam

    Additive manufacturing technologies are increasingly being used to replace standard extrusion or molding methods in engineering polymeric biomedical implants, which can be further seeded with cells for tissue regeneration. The principal advantage of this new technology is the ability to print directly from a scan and hence produce parts which are an ideal fit for an individual, eliminating much of the sizing and fitting associated with standard manufacturing methods. The question though arises whether devices which may be macroscopically similar, serve identical functions and are produced from the same material, interact in the same manner with cells and living tissue. Here we show that fundamental differences can exist between 3-D printed and extruded scaffolds which can impact stem cell differentiation and lineage selection. We will show how polymer confinement inherent in these methods affect the printed features on multiple length scales. We will also and how the differentiation of stem cells is affected by substrate heterogeneity in both morphological and mechanical features. NSF-Inspire award # 1344267.

  10. Antibody-free isolation of rare cancer cells from blood based on 3D lateral dielectrophoresis.

    Cheng, I-Fang; Huang, Wei-Lun; Chen, Tzu-Ying; Liu, Chien-Wei; Lin, Yu-De; Su, Wu-Chou

    2015-07-21

    We present an antibody-free approach for the high-purity and high-throughput dielectrophoretic (DEP) isolation of circulating tumour cells (CTCs) from blood in a microfluidic chip. A hydrodynamic sheath flow is designed upstream in the chip to direct the suspension samples to the channel side walls, thus providing a queue to allow DEP-induced lateral displacements. High-throughput continuous cancer cell sorting (maximum flow rate: ~2.4 mL h(-1), linear velocity: ~4 mm s(-1)) is achieved with a sustained 3D lateral DEP (LDEP) particle force normal to the continuous through-flow. This design allows the continuous fractionation of micro/nanosized particles into different downstream subchannels based on the differences in their different critical negative DEP strengths/mobilities. The main advantage of this separation strategy is that increasing the channel length can effectively increase the throughput proportionally. The effective separation of rare cancer cells (<0.001%) from diluted human blood in a handheld chip is demonstrated. An enrichment factor of 10(5) and a recovery rate of ~85% from a 0.001% cancer cell sample are achieved at an optimal flow rate of 20 μL min(-1) passing through a 6 cm long LDEP channel with an appropriate voltage at a frequency of 10 kHz. A higher throughput of 2.4 mL h(-1) is also achieved with a 13 cm long metal-based microchannel. PMID:26085231

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

    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.

  12. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation.

    Zhou, Xuan; Castro, Nathan J; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-01-01

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm(2) intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application. PMID:27597635

  13. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells

    Alain Carpentier

    2012-06-01

    Full Text Available Electrostimulation (ES can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 x 25 x 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002 and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01. Immunocytochemistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and bio- chemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases.

  14. Improving organic tandem solar cells based on water-processed nanoparticles by quantitative 3D nanoimaging.

    Pedersen, E B L; Angmo, D; Dam, H F; Thydén, K T S; Andersen, T R; Skjønsfjell, E T B; Krebs, F C; Holler, M; Diaz, A; Guizar-Sicairos, M; Breiby, D W; Andreasen, J W

    2015-08-28

    Organic solar cells have great potential for upscaling due to roll-to-roll processing and a low energy payback time, making them an attractive sustainable energy source for the future. Active layers coated with water-dispersible Landfester particles enable greater control of the layer formation and easier access to the printing industry, which has reduced the use of organic solvents since the 1980s. Through ptychographic X-ray computed tomography (PXCT), we image quantitatively a roll-to-roll coated photovoltaic tandem stack consisting of one bulk heterojunction active layer and one Landfester particle active layer. We extract the layered morphology with structural and density information including the porosity present in the various layers and the silver electrode with high resolution in 3D. The Landfester particle layer is found to have an undesired morphology with negatively correlated top- and bottom interfaces, wide thickness distribution and only partial surface coverage causing electric short circuits through the layer. By top coating a polymer material onto the Landfester nanoparticles we eliminate the structural defects of the layer such as porosity and roughness, and achieve the increased performance larger than 1 V expected for a tandem cell. This study highlights that quantitative imaging of weakly scattering stacked layers of organic materials has become feasible by PXCT, and that this information cannot be obtained by other methods. In the present study, this technique specifically reveals the need to improve the coatability and layer formation of Landfester nanoparticles, thus allowing improved solar cells to be produced. PMID:26220159

  15. X-ray tomography: Biological cells in 3-D at better than 50 nm resolution

    Full text: X-ray microscopy can be used to image whole, hydrated, specimens with a spatial resolution 5-10 times better than that obtained using visible light microscopy. X-ray imaging at photon energies below the K- absorption edge of oxygen, referred to as the water window, exploits the strong natural contrast for organic material embedded in a mostly water matrix. With a transmission X-ray microscope using Fresnel zone plate optics, specimens up to 10 microns thick can be examined. The highest X-ray transmission in hydrated samples is obtained at a wavelength of 2.4 nm but, due to the low numerical aperture of zone plate lenses operated in st order diffraction mode the structures resolved are much larger than the X-ray wavelength. Because of the low NA of X-ray lenses (NA=0.05), combined with the effect of polychromatic illumination and a wavelength dependant focal length, the effective depth of ld is large (6-10 microns). The experiments presented here were performed at the Advanced Light Source using the full ld transmission X-ray microscope, XM-1. This microscope employs a bend magnet X-ray source and zone plate condenser and objective lenses. The condenser zone plate acts as a monochromator and the X-ray images are recorded directly on a cooled, back-thinned 1024x1024 pixel CCD camera. The sample holder was a rotationally symmetric glass tube; the region containing the sample was 10 microns in diameter with a wall thickness of 200 nm. Live yeast cells were loaded into the tube, rapidly frozen by a blast of liquid nitrogen-cooled helium gas, and maintained at 140 deg C by a steady flow of cold helium. The image sequence spanned 180 deg and consisted of 45 images spaced by 4 deg. The images were aligned to a common axis and computed tomographic reconstruction was used to obtain the 3-D X-ray linear absorption coefficient. Volume rendering and animation of reconstructed data was performed using the 3-D program, Amira. Acquisition time for 90 images was 3 min

  16. Bacterial cell culture

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  17. Mammalian Cell Culture Simplified.

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  18. Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model.

    Takagi, Ryoji; Ishimaru, Junko; Sugawara, Ayaka; Toyoshima, Koh-Ei; Ishida, Kentaro; Ogawa, Miho; Sakakibara, Kei; Asakawa, Kyosuke; Kashiwakura, Akitoshi; Oshima, Masamitsu; Minamide, Ryohei; Sato, Akio; Yoshitake, Toshihiro; Takeda, Akira; Egusa, Hiroshi; Tsuji, Takashi

    2016-04-01

    The integumentary organ system is a complex system that plays important roles in waterproofing, cushioning, protecting deeper tissues, excreting waste, and thermoregulation. We developed a novel in vivo transplantation model designated as a clustering-dependent embryoid body transplantation method and generated a bioengineered three-dimensional (3D) integumentary organ system, including appendage organs such as hair follicles and sebaceous glands, from induced pluripotent stem cells. This bioengineered 3D integumentary organ system was fully functional following transplantation into nude mice and could be properly connected to surrounding host tissues, such as the epidermis, arrector pili muscles, and nerve fibers, without tumorigenesis. The bioengineered hair follicles in the 3D integumentary organ system also showed proper hair eruption and hair cycles, including the rearrangement of follicular stem cells and their niches. Potential applications of the 3D integumentary organ system include an in vitro assay system, an animal model alternative, and a bioengineered organ replacement therapy. PMID:27051874

  19. Pin cell discontinuity factors in the transient 3-D discrete ordinates code TORT-TD

    Even with the rapid increase of computing power, whole core transient and accident analyses based on the direct solution of the 3-D neutron transport equation with a large number of energy groups and a detailed heterogeneous description of the core still remain computationally challenging. Current industrial methods for reactor core calculations therefore involve a two step approach in which lattice (assembly) depletion transport methods are used to prepare energy collapsed and fuel assembly or pin cell homogenized cross sections for subsequent whole core transport calculations. Spatial homogenization, in principal, requires the knowledge of both the actual boundary condition (local core environment) of the fuel assembly and the exact heterogeneous flux distribution (reference solution) of the whole core problem within that fuel assembly. Since, in particular, the latter is not known a priori, an infinite medium (zero net current) condition is used in the lattice calculations. It is well known that this approximation may lead to undesirable errors in cores in which large flux gradients are present across the fuel assemblies. This is the case in cores that have high heterogeneity and/or strong local absorbers, e.g. PWRs with partial MOX loading and inserted control rod clusters. There are two major approaches to mitigate spatial homogenization errors, superhomogenization (SPH) factors, and discontinuity factors within the scope of equivalence theory (ET) and generalized equivalence theory (GET). Although discontinuity factors are usually applied at the level of fuel assembly node size (assembly discontinuity factors, ADF), the methodology can be extended to pin cell homogenized whole core calculations involving pin cell discontinuity factors (PDF). There are also further developments for both the diffusion and the simplified transport (SP3) equation. In this paper, PDFs are introduced into the time-dependent 3-D discrete ordinates code TORT-TD in order to reduce the

  20. Fish Stem Cell Cultures

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Full Text Available Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  1. Controlling the diversity of cell populations in a stem cell culture

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  2. Characterization of novel tumor stroma markers identified by gene expression profiling of human cancer tissues and 3D co-culture models

    The tumor stroma plays an important role in tumorigenesis. During cancer progression it undergoes changes in architecture, gene expression and secretion of proteolytic enzymes that are essential for the invasive and metastatic phenotype of malignant tumors. Cancer associated fibroblasts (Cafes) represent the major cellular component of the stroma and recent studies demonstrated the prognostic and therapeutic significance of CaF-related molecular signatures. The identification and characterization of genes and signaling pathways involved in the molecular interactions between tumor and stromal cells has been the focus of this study. For that purpose we have used two complementary approaches: the identification of novel tumor stroma targets in human colon cancer samples using whole genome Affymetrix GeneChip analysis and the validation of theses targets in a newly established of 3D co-culture model that mimics the cellular and molecular heterogeneity of human cancers. We have demonstrated increased expression of gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and TGFβ pathway activation in CAFs vs their normal counterparts in both systems. The putative TGFβ target IGFBP7 (insulin-like growth factor binding protein 7) was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. IGFPB7 was shown to promote anchorage-independent growth in malignant mesenchymal cells and malignant epithelial cells with an EMT-phenotype, whereas a tumor suppressor function was observed in tumor epithelial cells. In summary, we have demonstrated that a number of important signaling pathways involved in cancer progression and metastasis are specifically dysregulated in the tumor stroma both in our in vivo screen and in the in vitro 3D model, illustrating the value of these approaches for the identification and characterization of novel stromal markers. (author)

  3. Functional 3D Neural Mini-Tissues from Printed Gel-Based Bioink and Human Neural Stem Cells.

    Gu, Qi; Tomaskovic-Crook, Eva; Lozano, Rodrigo; Chen, Yu; Kapsa, Robert M; Zhou, Qi; Wallace, Gordon G; Crook, Jeremy M

    2016-06-01

    Direct-write printing of stem cells within biomaterials presents an opportunity to engineer tissue for in vitro modeling and regenerative medicine. Here, a first example of constructing neural tissue by printing human neural stem cells that are differentiated in situ to functional neurons and supporting neuroglia is reported. The supporting biomaterial incorporates a novel clinically relevant polysaccharide-based bioink comprising alginate, carboxymethyl-chitosan, and agarose. The printed bioink rapidly gels by stable cross-linking to form a porous 3D scaffold encapsulating stem cells for in situ expansion and differentiation. Differentiated neurons form synaptic contacts, establish networks, are spontaneously active, show a bicuculline-induced increased calcium response, and are predominantly gamma-aminobutyric acid expressing. The 3D tissues will facilitate investigation of human neural development, function, and disease, and may be adaptable for engineering other 3D tissues from different stem cell types. PMID:27028356

  4. Optimizing stem cell culture.

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-01-01

    International audience Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such a...

  5. Digital Microfluidic Cell Culture.

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  6. Mesenchymal stem cells enhance ovarian cancer cell infiltration through IL6 secretion in an amniochorionic membrane based 3D model

    Touboul Cyril

    2013-01-01

    Full Text Available Abstract Background The early peritoneal invasion of epithelial ovarian cancer (EOC by tumoral aggregates presents in ascites is a major concern. The role of the microenvironment seems to be important in this process but the lack of adequate models to study cellular interactions between cancer cells and stromal cells does not allow to uncover the molecular pathways involved. Our goal was to study the interactions between ovarian cancer cells (OCC and mesenchymal stem cells (MSC using a 3D model. Methods We used millimetric pieces of amniochorionic membrane - referred to as amniotic membrane scaffold (AMS - to create 3D peritoneal nodules mimicking EOC early invasion. We were able to measure the distribution and the depth of infiltration using confocal microsopy. We extracted MSC from the amniochorionic membrane using the markers CD34-, CD45-, CD73+, CD90+, CD105+ and CD29+ at the Fluorescence Activated Cell Sorting (FACS analysis. We used transwell and wound healing tests to test OCC migration and invasion in vitro. Results Here we show that OCC tumors were located in regions rich in MSC (70%. The tumors infiltrated deeper within AMS in regions rich in MSC (p Conclusions The use of tridimensional models using AMS could be a useful tool to decipher early molecular events in ovarian cancer metastasis. Cytokine inhibitors interrupting the cross-talk between OCCs and MSCs such as IL6 should be investigated as a new therapeutic approach in ovarian cancer.

  7. Cast and 3D printed ion exchange membranes for monolithic microbial fuel cell fabrication

    Philamore, Hemma; Rossiter, Jonathan; Walters, Peter; Winfield, Jonathan; Ieropoulos, Ioannis

    2015-09-01

    We present novel solutions to a key challenge in microbial fuel cell (MFC) technology; greater power density through increased relative surface area of the ion exchange membrane that separates the anode and cathode electrodes. The first use of a 3D printed polymer and a cast latex membrane are compared to a conventionally used cation exchange membrane. These new techniques significantly expand the geometric versatility available to ion exchange membranes in MFCs, which may be instrumental in answering challenges in the design of MFCs including miniaturisation, cost and ease of fabrication. Under electrical load conditions selected for optimal power transfer, peak power production (mean 10 batch feeds) was 11.39 μW (CEM), 10.51 μW (latex) and 0.92 μW (Tangoplus). Change in conductivity and pH of anolyte were correlated with MFC power production. Digital and environmental scanning electron microscopy show structural changes to and biological precipitation on membrane materials following long term use in an MFC. The cost of the novel membranes was lower than the conventional CEM. The efficacy of two novel membranes for ion exchange indicates that further characterisation of these materials and their fabrication techniques, shows great potential to significantly increase the range and type of MFCs that can be produced.

  8. Digital holography as a method for 3D imaging and estimating the biovolume of motile cells.

    Merola, F; Miccio, L; Memmolo, P; Di Caprio, G; Galli, A; Puglisi, R; Balduzzi, D; Coppola, G; Netti, P; Ferraro, P

    2013-12-01

    Sperm morphology is regarded as a significant prognostic factor for fertilization, as abnormal sperm structure is one of the most common factors in male infertility. Furthermore, obtaining accurate morphological information is an important issue with strong implications in zoo-technical industries, for example to perform sorting of species X from species Y. A challenging step forward would be the availability of a fast, high-throughput and label-free system for the measurement of physical parameters and visualization of the 3D shape of such biological specimens. Here we show a quantitative imaging approach to estimate simply and quickly the biovolume of sperm cells, combining the optical tweezers technique with digital holography, in a single and integrated set-up for a biotechnology assay process on the lab-on-a-chip scale. This approach can open the way for fast and high-throughput analysis in label-free microfluidic based "cytofluorimeters" and prognostic examination based on sperm morphology, thus allowing advancements in reproductive science. PMID:24129638

  9. Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

    Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters upon activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments

  10. Prospective use of the 3D printing technology for the microstructural engineering of Solid Oxide Fuel Cell components

    Hernandez-Rodriguez, E. M.; Acosta-Mora, P.; Mendez-Ramos, J.; Borges Chinea, E.; Esparza Ferrera, P.; Canales-Vazquez, J.; Nunez, P.; Ruiz-Morales, J.

    2014-07-01

    A cost-effective micro-manufacturing process to accurately build 3D microstructures for their prospective use in the fabrication of Solid Oxide Fuel Cells components has been tested. The 3D printing method, based on the stereo lithography, allows solidifying layer by layer a dispersion of ceramic material in a liquid photosensitive organic monomer. A simple projector, a computer-controlled z-stage and a few PowerPoint slides may be used for the fabrication of a wide range of complex 3D microstructures in few minutes. In this work, 3D ceramic microstructures based on the yttria-stabilized zirconia (YSZ) were successfully fabricated. The micro structured ceramic components produced were stable after sintering at 1400 degree centigrade for 4 h. Impedance measurements show that the fabrication process does not have any detrimental effect on the electrical properties of the structured material. (Author)

  11. Prospective use of the 3D printing technology for the microstructural engineering of Solid Oxide Fuel Cell components

    A cost-effective micro-manufacturing process to accurately build 3D microstructures for their prospective use in the fabrication of Solid Oxide Fuel Cells components has been tested. The 3D printing method, based on the stereo lithography, allows solidifying layer by layer a dispersion of ceramic material in a liquid photosensitive organic monomer. A simple projector, a computer-controlled z-stage and a few PowerPoint slides may be used for the fabrication of a wide range of complex 3D microstructures in few minutes. In this work, 3D ceramic microstructures based on the yttria-stabilized zirconia (YSZ) were successfully fabricated. The micro structured ceramic components produced were stable after sintering at 1400 degree centigrade for 4 h. Impedance measurements show that the fabrication process does not have any detrimental effect on the electrical properties of the structured material. (Author)

  12. 3D simulation as a tool for improving the safety culture during remediation work at Andreeva Bay

    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. (paper)

  13. 3D simulation as a tool for improving the safety culture during remediation work at Andreeva Bay.

    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

  14. Detection and analysis of human serum albumin nanoparticles within phagocytic cells at the resolution of individual live cell or single 3D multicellular spheroid

    Since nanoparticles (NPs) have shown great potential in various biomedical applications, live cell response to NPs should be thoroughly explored prior to their in vivo use. In the current study, live cell array (LCA) methodology and unique cell-based assays were used to study the interaction of magnetite (HSA-Mag NP) loaded human serum albumin NPs with phagocytic cells. The LCA enabled cell culturing during HSA-Mag NP accumulation and monolayer or spheroid formation, concomitantly with on-line monitoring of NP internalization. These platforms were also utilized for imaging intercellular links between living cells preloaded with HSA-Mag NP in 2D and 3D resolution. HSA-Mag NP uptake by cells was quantified by imaging, and analyzed using time-resolved measurements. Image analysis of the individual cells in cell populations showed accumulation of HSA-Mag NP by promonocytes and glial cells in a dose- and time-dependent manner. High variability of NP accumulation in individual cells within cell populations, as well as cell subgroups, was evident in both cell types. Following 24 h interaction, uptake of HSA-Mag NP was about 10 times more efficient in glial cells than in activated promonocytes. The presented assays may facilitate detection and analysis of the amount of NPs within individual cells, as well as the rate of NP accumulation and processing in different subsets of living cells. Such data are crucial for estimating predicted drug dosage delivered by NPs, as well as to study possible mechanisms for NP interference with live cells

  15. Detection and analysis of human serum albumin nanoparticles within phagocytic cells at the resolution of individual live cell or single 3D multicellular spheroid

    Afrimzon, Elena; Zurgil, Naomi; Sobolev, Maria; Shafran, Yana [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel); Langer, Klaus; Zlatev, Iavor [Westfälischen Wilhelms-Universität Münster, Institut für Pharmazeutische Technologie und Biopharmazie (Germany); Wronski, Robert; Windisch, Manfred [QPS Austria GmbH (Austria); Briesen, Hagen von [Fraunhofer Institute for Biomedical Engineering IBMT, Department of Cell Biology & Applied Virology (Germany); Schmidt, Reinhold [Medical University of Graz, Department of Neurology (Austria); Pietrzik, Claus [University Medical Center of the Johannes Gutenberg University of Mainz, Institute of Pathobiochemistry (Germany); Deutsch, Mordechai, E-mail: motti.jsc@gmail.com [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel)

    2015-12-15

    Since nanoparticles (NPs) have shown great potential in various biomedical applications, live cell response to NPs should be thoroughly explored prior to their in vivo use. In the current study, live cell array (LCA) methodology and unique cell-based assays were used to study the interaction of magnetite (HSA-Mag NP) loaded human serum albumin NPs with phagocytic cells. The LCA enabled cell culturing during HSA-Mag NP accumulation and monolayer or spheroid formation, concomitantly with on-line monitoring of NP internalization. These platforms were also utilized for imaging intercellular links between living cells preloaded with HSA-Mag NP in 2D and 3D resolution. HSA-Mag NP uptake by cells was quantified by imaging, and analyzed using time-resolved measurements. Image analysis of the individual cells in cell populations showed accumulation of HSA-Mag NP by promonocytes and glial cells in a dose- and time-dependent manner. High variability of NP accumulation in individual cells within cell populations, as well as cell subgroups, was evident in both cell types. Following 24 h interaction, uptake of HSA-Mag NP was about 10 times more efficient in glial cells than in activated promonocytes. The presented assays may facilitate detection and analysis of the amount of NPs within individual cells, as well as the rate of NP accumulation and processing in different subsets of living cells. Such data are crucial for estimating predicted drug dosage delivered by NPs, as well as to study possible mechanisms for NP interference with live cells.

  16. Immunogenicity and T cell recognition in swine of foot-and-mouth disease virus polymerase 3D

    Immunization of domestic pigs with a vaccinia virus (VV) recombinant expressing foot-and-mouth disease virus (FMDV) 3D protein conferred partial protection against challenge with infectious virus. The severity reduction of the clinical symptoms developed by the challenged animals occurred in the absence of significant levels of anti-3D circulating antibodies. This observation suggested that the partial protection observed was mediated by the induction of a 3D-specific cellular immune response. To gain information on the T cell recognition of FMDV 3D protein, we conducted in vitro proliferative assays using lymphocytes from outbred pigs experimentally infected with FMDV and 90 overlapping peptides spanning the complete 3D sequence. The use of pools of two to three peptides allowed the identification of T cell epitopes that were efficiently recognized by lymphocytes from at least four of the five animals analyzed. This recognition was heterotypic because anti-peptide responses increased upon reinfection of animals with a FMDV isolate from a different serotype. The results obtained with individual peptides confirmed the antigenicity observed with peptide pools. Detection of cytokine mRNAs by RT-PCR in lymphocytes stimulated in vitro by individual 3D peptides revealed that IFN-γ mRNA was the most consistently induced, suggesting that the activated T cells belong to the Th 1 subset. These results indicate that 3D protein contains epitopes that can be efficiently recognized by porcine T lymphocytes from different infected animals, both upon primary and secondary (heterotypic) FMDV infection. These epitopes can extend the repertoire of viral T cell epitopes to be included in subunit and synthetic FMD vaccines

  17. From Three-Dimensional Cell Culture to Organs-on-Chips

    Huh, Dongeun; Hamilton, Geraldine A.; Ingber, Donald E.

    2011-01-01

    Three-dimensional (3D) cell culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional two-dimensional (2D) culture systems. Here, we review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue inter...

  18. Mycoplasmas detection in cells cultures

    Rivera-Tapia José Antonio; Castillo-Viveros Linda Valeria; Sánchez-Hernández José Antonio

    2010-01-01

    INTRODUCTION. Cells cultures are widely used in both biomedical and biotechnological research centers and industry, as well as for diagnostic test in hospitals. Contaminations of cells cultures with microbial organisms as well as with virus or other eukaryotic cell lines are a major problem in cell culture related research.OBJECTIVE. Mycoplasmas detection in cells cultures came from biomedical laboratories.MATERIAL AND METHODS. The cells cultures screened for mycoplasmas by using of microbiol...

  19. Perfusion based cell culture chips

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and ti...

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

    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

  1. 1-integrin and MT1-MMP promote tumor cell migration in 2D but not in 3D fibronectin microenvironments

    Corall, Silke; Haraszti, Tamas; Bartoschik, Tanja; Spatz, Joachim Pius; Ludwig, Thomas; Cavalcanti-Adam, Elisabetta Ada

    2014-03-01

    Cell migration is a crucial event for physiological processes, such as embryonic development and wound healing, as well as for pathological processes, such as cancer dissemination and metastasis formation. Cancer cell migration is a result of the concerted action of matrix metalloproteinases (MMPs), expressed by cancer cells to degrade the surrounding matrix, and integrins, the transmembrane receptors responsible for cell binding to matrix proteins. While it is known that cell-microenvironment interactions are essential for migration, the role of the physical state of such interactions remains still unclear. In this study we investigated human fibrosarcoma cell migration in two-dimensional (2D) and three-dimensional (3D) fibronectin (FN) microenvironments. By using antibody blocking approach and cell-binding site mutation, we determined that -integrin is the main mediator of fibrosarcoma cell migration in 2D FN, whereas in 3D fibrillar FN, the binding of - and -integrins is not necessary for cell movement in the fibrillar network. Furthermore, while the general inhibition of MMPs with GM6001 has no effect on cell migration in both 2D and 3D FN matrices, we observed opposing effect after targeted silencing of a membrane-bound MMP, namely MT1-MMP. In 2D fibronectin, silencing of MT1-MMP results in decreased migration speed and loss of directionality, whereas in 3D FN matrices, cell migration speed is increased and integrin-mediated signaling for actin dynamics is promoted. Our results suggest that the fibrillar nature of the matrix governs the migratory behavior of fibrosarcoma cells. Therefore, to hinder migration and dissemination of diseased cells, matrix molecules should be directly targeted, rather than specific subtypes of receptors at the cell membrane.

  2. Synthesis of TiO2 nanotube arrays and its application in mini-3D dye-sensitized solar cells

    Here we report a mini-three-dimensional dye-sensitized solar cell (3D DSSC) based on TiO2 nanotube arrays (TNAs). TNAs were directly grown on spiral-shaped titanium wire via a facile potentiostatic anodization. Furthermore, the TNAs film was characterized by scanning electron microscopy, x-ray diffraction and transmission electron microscopy, indicating that the annealed TNAs were composed of single-crystalline anatase particles. Unlike conventional flat DSSC, this mini-3D DSSC could easily hold liquid electrolyte due to the capillary force which facilitated sealing the cell. This mini-3D DSSC showed an energy conversion efficiency of 4.1% under the AM 1.5 condition, which was much higher compared with that (3.2%) of the backside illuminated TNAs based DSSC of the same projected area.

  3. 3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

    Hummel, Eric; Guttmann, Peter; Werner, Stephan; Tarek, Basel; SCHNEIDER, Gerd; Kunz, Michael; Frangakis, Achilleas S.; Westermann, Benedikt

    2012-01-01

    The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial re...

  4. 3D ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography

    Hummel, Eric; Guttmann, Peter; Werner, Stephan; Tarek, Basel; SCHNEIDER, Gerd; Kunz, Michael; Frangakis, Achilleas S.; Westermann, Benedikt

    2012-01-01

    The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial re...

  5. The effect of porosity on cell ingrowth into accurately defined, laser-made, polylactide-based 3D scaffolds

    Danilevicius, Paulius; Georgiadi, Leoni [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Pateman, Christopher J.; Claeyssens, Frederik [Kroto Research Institute, Department of Materials Science and Engineering, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom); Chatzinikolaidou, Maria, E-mail: mchatzin@materials.uoc.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Department of Materials Science and Technology, University of Crete, PO Box 2208, 71303 Heraklion (Greece); Farsari, Maria, E-mail: mfarsari@iesl.forth.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece)

    2015-05-01

    Highlights: • We studied the porosity of laser-made 3D scaffolds on MC3T3-E1 pre-osteoblastic cells. • We made polylactide 3D scaffolds with pores 25–110 μm. - Abstract: The aim of this study is to demonstrate the accuracy required for the investigation of the role of solid scaffolds’ porosity in cell proliferation. We therefore present a qualitative investigation into the effect of porosity on MC3T3-E1 pre-osteoblastic cell ingrowth of three-dimensional (3D) scaffolds fabricated by direct femtosecond laser writing. The material we used is a purpose made photosensitive pre-polymer based on polylactide. We designed and fabricated complex, geometry-controlled 3D scaffolds with pore sizes ranging from 25 to 110 μm, representing porosities 70%, 82%, 86%, and 90%. The 70% porosity scaffolds did not support cell growth initially and in the long term. For the other porosities, we found a strong adhesion of the pre-osteoblastic cells from the first hours after seeding and a remarkable proliferation increase after 3 weeks and up to 8 weeks. The 86% porosity scaffolds exhibited a higher efficiency compared to 82% and 90%. In addition, bulk material degradation studies showed that the employed, highly-acrylated polylactide is degradable. These findings support the potential use of the proposed material and the scaffold fabrication technique in bone tissue engineering.

  6. Designing a binding interface for control of cancer cell adhesion via 3D topography and metabolic oligosaccharide engineering.

    Du, Jian; Che, Pao-Lin; Wang, Zhi-Yun; Aich, Udayanath; Yarema, Kevin J

    2011-08-01

    This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three-dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac(5)ManNTGc, a thiol-bearing analog of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bio-orthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion. PMID:21549424

  7. Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering.

    Parvizi, Mojtaba; Bolhuis-Versteeg, Lydia A M; Poot, André A; Harmsen, Martin C

    2016-07-01

    Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep-free poly (1,3-trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue-derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF-β1 for 7d. Phenotype and function were assessed by qRT-PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non-differentiated and pre-differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT-PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF-β1-driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre-differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well-aligned ASC-derived SMC which deposited ECM. Under the same conditions, pre-differentiated ASC-derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF-β1 pre-stimulation to generate SMC from ASC and that pre-differentiated ASC keep their SMC phenotype with increased expression of SMC markers. PMID:26989865

  8. 3D Ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography.

    Eric Hummel

    Full Text Available The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.

  9. New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells

    Rowehl Rebecca A

    2010-07-01

    Full Text Available Abstract Background Growing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133high/CD44high fraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity in vivo and in vitro, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133high/CD44high colon cancer cells; and b to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214. Results Selected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (SABiosciences revealed that colonospheres induced by purified CD133high/CD44high expressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 μM for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and

  10. Particle optics in the TIT-RFQ calculated using a 3D particle-in-cell code

    Beam dynamics in an RFQ at the Tokyo Institute of Technology was analyzed using a 3D particle-in-cell computer code. In this calculation not only space charge force between each macroparticles but also 3D image charge field were included. Beam transmission performance was calculated for two types of vane-tip design with different tip curvature radii. These results are compared with ones obtained with the idealized linear two-term potential. The old vane tip design with a small tip curvature radius has given very poor beam transmission efficiency which cannot be accepted for the actual machine. (author)

  11. Dose distribution of IMRT and 3D-CRT on treating central non-small-cell lung cancer

    3D-CRT and IMRT were used in the radiation therapy of Central Non-small-cell lung cancer (NSCLC), and the dose difference of the methods was estimated. Thirty-two patients suffering with II class NSCLC were selected. Based on CT images, each patient was given 1 3D-CRT (3 dimensional conformal radiotherapy) and 2 IMRT(intensity modulated radiation therapy) treatment plans (5 fields and 7 fields), respectively, and the dose distribution was evaluated too. The results showed that PTVDmean and the PTVmax, PTVDmax (%) and CI of IMRT were both higher than those of 3D-CRT, but the uniformity was not as good as 3D-CRT. All indexes of lung and spinal cord treated with IMRT were lower than that treated with 3D-CRT. Moreover, there was no significance of the difference between 5 fields and 7 fields. In a conclusion, IMRT could not only decrease the target dose of NSCLC, but it can protect normal tissue from radiation damage effectively. And when IMRT was used, 5 fields might be enough. (authors)

  12. Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone) Scaffolds.

    Palomeras, Sònia; Rabionet, Marc; Ferrer, Inés; Sarrats, Ariadna; Garcia-Romeu, Maria Luisa; Puig, Teresa; Ciurana, Joaquim

    2016-01-01

    The cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs' phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone) (PCL), a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population. PMID:27120585

  13. Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone Scaffolds

    Sònia Palomeras

    2016-04-01

    Full Text Available The cancer stem cell (CSC population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs’ phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone (PCL, a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control. Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population.

  14. 3D Plant Cell Architecture of Arabidopsis thaliana (Brassicaceae Using Focused Ion Beam–Scanning Electron Microscopy

    Bhawana

    2014-06-01

    Full Text Available Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies.

  15. From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy.

    Coralie Spiegelhalter

    Full Text Available BACKGROUND: In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM to electron microscopy (EM. METHODOLOGY: To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1 phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1 expression. CONCLUSION: Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.

  16. Development of non-contact 3D measurement system for HALF cells

    We strive to develop a 3D coordinate measuring machine, which can measure the shape of parts of accelerator cavity with without contact and rapidly. Currently, the ILC (International Linear Collider) project is progressing through international collaboration. The major goal of ILC is to produce and investigate Higgs bosons. ILC consists of two linear accelerators facing each other, and will hurl some 10 billion electrons and positrons toward each other at nearly the speed of light. The cavity is an important component to accelerate particles to near light speed. A cavity's inner 3D shape influences the accelerating performance. Therefore, it is important to measure the shape of the parts of a cavity. We are developing a highly accurate and non-contact shape measuring machine using triangulation method. (author)

  17. Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells

    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 extracellular 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 versus 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.

  18. Characterizing microscale aluminum composite layer properties on silicon solar cells with hybrid 3D scanning force measurements

    Bae, Sung-Kuk; Choi, Beomjoon; Chung, Haseung; Shin, Seungwon; Song, Hee-Eun; Seo, Jung Hwan

    2016-03-01

    This article presents a novel technique to estimate the mechanical properties of the aluminum composite layer on silicon solar cells by using a hybrid 3-dimensional laser scanning force measurement (3-D LSFM) system. The 3-D LSFM system measures the material properties of sub-layers constituting a solar cell. This measurement is critical for realizing high-efficient ultra-thin solar cells. The screen-printed aluminum layer, which significantly affects the bowing phenomenon, is separated from the complete solar cell by removing the silicon (Si) layer with deep reactive ion etching. An elastic modulus of ~15.1 GPa and a yield strength of ~35.0 MPa for the aluminum (Al) composite layer were obtained by the 3-D LSFM system. In experiments performed for 6-inch Si solar cells, the bowing distances decreased from 12.02 to 1.18 mm while the Si layer thicknesses increased from 90 to 190 μm. These results are in excellent agreement with the theoretical predictions for ultra-thin Si thickness (90 μm) based on the obtained Al composite layer properties.

  19. Microfluidic Cell Culture Device

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

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

    Giovanna Sansoni

    2009-01-01

    Full Text Available 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.

  1. Evaluation of anti-HER2 scFv-conjugated PLGA-PEG nanoparticles on 3D tumor spheroids of BT474 and HCT116 cancer cells

    Thuy Duong Le, Thi; Pham, Thu Hong; Nghia Nguyen, Trong; Giang Ngo, Thi Hong; Nhung Hoang, Thi My; Huan Le, Quang

    2016-06-01

    Three-dimensional culture cells (spheroids) are one of the multicellular culture models that can be applied to anticancer chemotherapeutic development. Multicellular spheroids more closely mimic in vivo tumor-like patterns of physiologic environment and morphology. In previous research, we designed docetaxel-loaded pegylated poly(D, L-lactide-co-glycolide) nanoparticles conjugated with anti-HER2 single chain antibodies (scFv-Doc-PLGA-PEG) and evaluated them in 2D cell culture. In this study, we continuously evaluate the cellular uptake and cytotoxic effect of scFv-Doc-PLGA-PEG on a 3D tumor spheroid model of BT474 (HER2-overexpressing) and HCT116 (HER2-underexpressing) cancer cells. The results showed that the nanoparticle formulation conjugated with scFv had a significant internalization effect on the spheroids of HER2-overexpressing cancer cells as compared to the spheroids of HER2-underexpressing cancer cells. Therefore, cytotoxic effects of targeted nanoparticles decreased the size and increased necrotic score of HER2-overexpressing tumor spheroids. Thus, these scFv-Doc-PLGA-PEG nanoparticles have potential for active targeting for HER2-overexpressing cancer therapy. In addition, BT474 and HCT116 spheroids can be used as a tumor model for evaluation of targeting therapies.

  2. Dynamic heterogeneity of DNA methylation and hydroxymethylation in embryonic stem cell populations captured by single-cell 3D high-content analysis

    Tajbakhsh, Jian, E-mail: tajbakhshj@cshs.org [Chromatin Biology Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Stefanovski, Darko [Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19348 (United States); Tang, George [Chromatin Biology Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Wawrowsky, Kolja [Translational Cytomics Group, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Liu, Naiyou; Fair, Jeffrey H. [Department of Surgery and UF Health Comprehensive Transplant Center, University of Florida College of Medicine, Gainesville, FL 32608 (United States)

    2015-03-15

    Cell-surface markers and transcription factors are being used in the assessment of stem cell fate and therapeutic safety, but display significant variability in stem cell cultures. We assessed nuclear patterns of 5-hydroxymethylcytosine (5hmC, associated with pluripotency), a second important epigenetic mark, and its combination with 5-methylcytosine (5mC, associated with differentiation), also in comparison to more established markers of pluripotency (Oct-4) and endodermal differentiation (FoxA2, Sox17) in mouse embryonic stem cells (mESC) over a 10-day differentiation course in vitro: by means of confocal and super-resolution imaging together with 3D high-content analysis, an essential tool in single-cell screening. In summary: 1) We did not measure any significant correlation of putative markers with global 5mC or 5hmC. 2) While average Oct-4 levels stagnated on a cell-population base (0.015 lnIU/day), Sox17 and FoxA2 increased 22-fold and 3-fold faster, respectively (Sox17: 0.343 lnIU/day; FoxA2: 0.046 lnIU/day). In comparison, global DNA methylation levels increased 4-fold faster (0.068 lnIU/day), and global hydroxymethylation declined at 0.046 lnIU/day, both with a better explanation of the temporal profile. 3) This progression was concomitant with the occurrence of distinct nuclear codistribution patterns that represented a heterogeneous spectrum of states in differentiation; converging to three major coexisting 5mC/5hmC phenotypes by day 10: 5hmC{sup +}/5mC{sup −}, 5hmC{sup +}/5mC{sup +}, and 5hmC{sup −}/5mC{sup +} cells. 4) Using optical nanoscopy we could delineate the respective topologies of 5mC/5hmC colocalization in subregions of nuclear DNA: in the majority of 5hmC{sup +}/5mC{sup +} cells 5hmC and 5mC predominantly occupied mutually exclusive territories resembling euchromatic and heterochromatic regions, respectively. Simultaneously, in a smaller subset of cells we observed a tighter colocalization of the two cytosine variants, presumably

  3. Dynamic heterogeneity of DNA methylation and hydroxymethylation in embryonic stem cell populations captured by single-cell 3D high-content analysis

    Cell-surface markers and transcription factors are being used in the assessment of stem cell fate and therapeutic safety, but display significant variability in stem cell cultures. We assessed nuclear patterns of 5-hydroxymethylcytosine (5hmC, associated with pluripotency), a second important epigenetic mark, and its combination with 5-methylcytosine (5mC, associated with differentiation), also in comparison to more established markers of pluripotency (Oct-4) and endodermal differentiation (FoxA2, Sox17) in mouse embryonic stem cells (mESC) over a 10-day differentiation course in vitro: by means of confocal and super-resolution imaging together with 3D high-content analysis, an essential tool in single-cell screening. In summary: 1) We did not measure any significant correlation of putative markers with global 5mC or 5hmC. 2) While average Oct-4 levels stagnated on a cell-population base (0.015 lnIU/day), Sox17 and FoxA2 increased 22-fold and 3-fold faster, respectively (Sox17: 0.343 lnIU/day; FoxA2: 0.046 lnIU/day). In comparison, global DNA methylation levels increased 4-fold faster (0.068 lnIU/day), and global hydroxymethylation declined at 0.046 lnIU/day, both with a better explanation of the temporal profile. 3) This progression was concomitant with the occurrence of distinct nuclear codistribution patterns that represented a heterogeneous spectrum of states in differentiation; converging to three major coexisting 5mC/5hmC phenotypes by day 10: 5hmC+/5mC−, 5hmC+/5mC+, and 5hmC−/5mC+ cells. 4) Using optical nanoscopy we could delineate the respective topologies of 5mC/5hmC colocalization in subregions of nuclear DNA: in the majority of 5hmC+/5mC+ cells 5hmC and 5mC predominantly occupied mutually exclusive territories resembling euchromatic and heterochromatic regions, respectively. Simultaneously, in a smaller subset of cells we observed a tighter colocalization of the two cytosine variants, presumably delineating chromatin domains in remodeling. We

  4. Improving organic tandem solar cells based on water-processed nanoparticles by quantitative 3D nanoimaging

    Pedersen, Emil Bøje Lind; Angmo, Dechan; Dam, Henrik Friis;

    2015-01-01

    particle active layer. We extract the layered morphology with structural and density information including the porosity present in the various layers and the silver electrode with high resolution in 3D. The Landfester particle layer is found to have an undesired morphology with negatively correlated top...... easier access to the printing industry, which has reduced the use of organic solvents since the 1980s. Through ptychographic X-ray computed tomography (PXCT), we image quantitatively a roll-to-roll coated photovoltaic tandem stack consisting of one bulk heterojunction active layer and one Landfester...

  5. Parallel 3-D particle-in-cell modelling of charged ultrarelativistic beam dynamics

    Boronina, Marina A.; Vshivkov, Vitaly A.

    2015-12-01

    > ) in supercolliders. We use the 3-D set of Maxwell's equations for the electromagnetic fields, and the Vlasov equation for the distribution function of the beam particles. The model incorporates automatically the longitudinal effects, which can play a significant role in the cases of super-high densities. We present numerical results for the dynamics of two focused ultrarelativistic beams with a size ratio 10:1:100. The results demonstrate high efficiency of the proposed computational methods and algorithms, which are applicable to a variety of problems in relativistic plasma physics.

  6. The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

    Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression

  7. The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

    Fujiwara, Daisuke [Department of Esophageal and Gastroenterological Surgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp [Department of Biomedical Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585 (Japan); Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Nohara, Shigeo; Iwanuma, Yoshimi; Kajiyama, Yoshiaki [Department of Esophageal and Gastroenterological Surgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2013-05-17

    Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression

  8. Epstein-Barr virus (EBV) infection of murine L cells expressing recombinant human EBV/C3d receptor.

    Ahearn, J M; Hayward, S D; Hickey, J C; Fearon, D T

    1988-01-01

    The normal host range of Epstein-Barr virus (EBV) is limited to primate B lymphocytes and certain epithelial cells that express the C3d/EBV receptor [complement receptor 2 (CR2, CD21)]. In the present study, expansion of the tissue tropism of EBV has been accomplished by stably transfecting the murine fibroblast L cell line with pMT.CR2. neo.1, a eukaryotic expression vector promoting the transcription of a complementary DNA insert encoding human CR2. High CR2-expressing transfected L cells w...

  9. Fully 3D Particle-in-Cell Simulation of Double Post-Hole Convolute on PTS Facility

    Zhao, Hailong; Dong, Ye; Zhou, Haijing; Zou, Wenkang; Institute of Fluid Physics Collaboration; Institute of Applied Physics; Computational Mathematics Collaboration

    2015-11-01

    In order to get better understand of energy transforming and converging process during High Energy Density Physics (HEDP) experiments, fully 3D particle-in-cell (PIC) simulation code NEPTUNE3D was used to provide numerical approach towards parameters which could hardly be acquired through diagnostics. Cubic region (34cm × 34cm × 18cm) including the double post-hole convolute (DPHC) on the primary test stand (PTS) facility was chosen to perform a series of fully 3D PIC simulations, calculating ability of codes were tested and preliminary simulation results about DPHC on PTS facility were discussed. Taking advantages of 3D simulation codes and large-scale parallel computation, massive data (~ 250GB) could be acquired in less than 5 hours and clear process of current transforming and electron emission in DPHC were demonstrated with the help of visualization tools. Cold-chamber tests were performed during which only cathode electron emission was considered without temperature rise or ion emission, current loss efficiency was estimated to be 0.46% ~ 0.48% by comparisons between output magnetic field profiles with or without electron emission. Project supported by the National Natural Science Foundation of China (Grant No. 11205145, 11305015, 11475155).

  10. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells

    Guo, Weibo; Wang, Shu; Yu, Xin; Qiu, Jichuan; Li, Jianhua; Tang, Wei; Li, Zhou; Mou, Xiaoning; Liu, Hong; Wang, Zhonglin

    2016-01-01

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells.The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold

  11. Cellular differentiation in 3D-bioprinted mesenchymal stem cell-loaded hydrogels with varying structural and mechanical properties

    Duarte Campos, Daniela Filipa

    2016-01-01

    Hydrogels are a promising alternative to rigid biomaterials typically used in the field of bone tissue engineering for the treatment of musculoskeletal disorders. By hydrogel-based 3D-bioprinting, the native ornamentation of cells and matrix from bone tissue could be resembled. Herein, it was hypothesized the combination of polysaccharides (agarose, alginate) with biological components (collagen, fibrinogen) would increase mechanical stiffness of printed constructs as well as support the prin...

  12. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C.; Wang, Lin

    2014-01-01

    Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approac...

  13. Test chambers for cell culture in static magnetic field

    Article presents a test chamber intended to be used for in vitro cell culture in homogenous constant magnetic field with parametrically variable magnitude. We constructed test chambers with constant parameters of control homeostasis of cell culture for the different parameters of static magnetic field. The next step was the computer calculation of 2D and 3D simulation of the static magnetic field distribution in the chamber. The analysis of 2D and 3D calculations of magnetic induction in the cells' exposition plane reveals, in comparison to the detection results, the greater accuracy of 2D calculations (Figs. 9 and 10). The divergence in 2D method was 2–4% and 8 to 10% in 3D method (reaching 10% only out of the cells′ cultures margins). -- Highlights: ► We present test chamber to be used for in vitro cell culture in static magnetic field. ► The technical data of the chamber construction was presented. ► 2D versus 3D simulation of static magnetic field distribution in chamber was reported. ► We report the accuracy of 2D calculation than 3D

  14. Double targeting of Survivin and XIAP radiosensitizes 3D grown human colorectal tumor cells and decreases migration

    Background and purpose: In the present study, we aimed to investigate the effect of single and double knockdown of the inhibitor of apoptosis proteins (IAP) Survivin and X-linked IAP (XIAP) on three-dimensional (3D) clonogenic survival, migration capacity and underlying signaling pathways. Materials and methods: Colorectal cancer cell lines (HCT-15, SW48, SW480, SW620) were subjected to siRNA-mediated single or Survivin/XIAP double knockdown followed by 3D colony forming assays, cell cycle analysis, Caspase activity assays, migration assays, matrigel transmigration assays and Western blotting (Survivin, XIAP, Focal adhesion kinase (FAK), p-FAK Y397, Akt1, p-Akt1 S473, Extracellular signal-regulated kinase (ERK1/2), p-ERK1/2 T202/Y204, Glycogen synthase kinase (GSK)3β, p-GSK3β S9, nuclear factor (NF)-κB p65). Results: While basal cell survival was altered cell line-dependently, Survivin or XIAP single and Survivin/XIAP double knockdown enhanced cellular radiosensitivity of all tested cancer cell lines grown in 3D. Particularly double knockdown conditions revealed accumulation of cells in G2/M, increased subG1 fraction, elevated Caspase 3/7 activity, and reduced migration. Intracellular signaling showed dephosphorylation of FAK and Akt1 upon Survivin and/or Survivin/XIAP silencing. Conclusions: Our results strengthen the notion of Survivin and XIAP to act as radiation resistance factors and further indicate that these apoptosis-regulating proteins are also functioning in cell cycling and cell migration

  15. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells.

    Guo, Weibo; Wang, Shu; Yu, Xin; Qiu, Jichuan; Li, Jianhua; Tang, Wei; Li, Zhou; Mou, Xiaoning; Liu, Hong; Wang, Zhonglin

    2016-01-21

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells. PMID:26750302

  16. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells.

    Drummond, Coyne G; Nickerson, Cheryl A; Coyne, Carolyn B

    2016-01-01

    Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the

  17. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

    Drummond, Coyne G.

    2015-01-01

    ABSTRACT Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and

  18. Automated quantitative analysis of 3D morphology and mean corpuscular hemoglobin in human red blood cells stored in different periods.

    Moon, Inkyu; Yi, Faliu; Lee, Yeon H; Javidi, Bahram; Boss, Daniel; Marquet, Pierre

    2013-12-16

    Quantitative phase (QP) images of red blood cells (RBCs), which are obtained by off-axis digital holographic microscopy, can provide quantitative information about three-dimensional (3D) morphology of human RBCs and the characteristic properties such as mean corpuscular hemoglobin (MCH) and MCH surface density (MCHSD). In this paper, we investigate modifications of the 3D morphology and MCH in RBCs induced by the period of storage time for the purpose of classification of RBCs with different periods of storage by using off-axis digital holographic microscopy. The classification of RBCs based on the duration of storage is highly relevant because a long storage of blood before transfusion may alter the functionality of RBCs and, therefore, cause complications in patients. To analyze any changes in the 3D morphology and MCH of RBCs due to storage, we use data sets from RBC samples stored for 8, 13, 16, 23, 27, 30, 34, 37, 40, 47, and 57 days, respectively. The data sets consist of more than 3,300 blood cells in eleven classes, with more than 300 blood cells per class. The classes indicate the storage period of RBCs and are listed in chronological order. Using the RBCs donated by healthy persons, the off-axis digital holographic microscopy reconstructs several quantitative phase images of RBC samples stored for eleven different periods. We employ marker-controlled watershed transform to remove the background in the RBC quantitative phase images obtained by the off-axis digital holographic microscopy. More than 300 single RBCs are extracted from the segmented quantitative phase images for each class. Such a large number of RBC samples enable us to obtain statistical distributions of the characteristic properties of RBCs after a specific period of storage. Experimental results show that the 3D morphology of the RBCs, in contrast to MCH, is essentially related to the aging of the RBCs. PMID:24514667

  19. 3D micro-XRF for cultural heritage objects: new analysis strategies for the investigation of the Dead Sea Scrolls.

    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

  20. Multiscale modeling of mechanosensing channels on vesicles and cell membranes in 3D constricted flows and shear flows

    Peng, Zhangli; Pak, On Shun; Young, Yuan-Nan; Liu, Allen; Stone, Howard

    2015-11-01

    We investigate the gating of mechanosensing channels (Mscls) on vesicles and cell membranes under different flow conditions using a multiscale approach. At the cell level (microns), the membrane tension is calculated using a 3D two-component whole-cell membrane model based on dissipative particle dynamics (DPD), including the cortex cytoskeleton and its interactions with the lipid bilayer. At the Mscl level (nanometers), we predict the relation between channel gating and the membrane tension obtained from a cell-level model using a semi-analytical model based on the bilayer hydrophobic mismatch energy. We systematically study the gating of Mscls of vesicles and cell membranes in constricted channel flows and shear flows, and explore the dependence of the gating on flow rate, cell shape and size. The results provide guidance for future experiments in inducing Mscl opening for various purposes such as drug delivery.

  1. Controlled positioning of cells in biomaterials - approaches towards 3D tissue printing

    Sandra Hofmann; Ralph Müller; Silke Wüst

    2011-01-01

    Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extrac...

  2. Poly(amido amine)-based multilayered thin films on 2D and 3D supports for surface-mediated cell transfection.

    Hujaya, Sry D; Marchioli, Giulia; Roelofs, Karin; van Apeldoorn, Aart A; Moroni, Lorenzo; Karperien, Marcel; Paulusse, Jos M J; Engbersen, Johan F J

    2015-05-10

    Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D. Therefore, COS-7 cells were cultured on top of multilayer films formed by layer-by-layer dipcoating of these polymers with GFP-encoded pDNA, and the effect of the number of layers and cell seeding density on the transfection efficiency was evaluated. Multilayer films with pCABOL were found to be superior to pCHIS in facilitating transfection, which was attributed to higher incorporation of pDNA and release of the transfection agent. High amounts of transfected cells were obtained on pCABOL films, correlating proportionally over a wide range with seeding density. Optimal transfection efficiency was obtained with pCABOL films composed of 10 bilayers. Further increase in the number of bilayers only marginally increased transfection efficiency. Using the optimal multilayer and cell seeding conditions, pCABOL multilayers were fabricated on poly(ε-caprolactone) (PCL), heparinized PCL (PCL-HEP), and poly(lactic acid) (PLA) disks as examples of common biomedical supports. The multilayers were found to completely mask the properties of the original substrates, with significant improvement in cell adhesion, which is especially pronounced for PCL and PLA disks. With all these substrates, transfection efficiency was found to be in the range of 25-50% transfected cells. The pCABOL/pDNA multilayer films can also conveniently add transfection capability to 3D scaffolds. Significant improvement in cell adhesion was observed after multilayer coating of 3D-plotted fibers of PCL (with and without an additional covalent heparin layer), especially for the PCL scaffold without heparin layer and transfection was observed on both 3D PCL and PCL-HEP scaffolds. These results show that layer

  3. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells

    Niemöller, Arvid; Jakes, Peter; Kayser, Steffen; Lin, Yu; Lehnert, Werner; Granwehr, Josef

    2016-08-01

    Electrochemical cells contain electrically conductive components, which causes various problems if such a cell is analyzed during operation in an EPR resonator. The optimum cell design strongly depends on the application and it is necessary to make certain compromises that need to be individually arranged. Rapid prototyping presents a straightforward option to implement a variable cell design that can be easily adapted to changing requirements. In this communication, it is demonstrated that sample containers produced by 3D printing are suitable for EPR applications, with a particular emphasis on electrochemical applications. The housing of a high temperature polymer electrolyte fuel cell (HT-PEFC) with a phosphoric acid doped polybenzimidazole membrane was prepared from polycarbonate by 3D printing. Using a custom glass Dewar, this fuel cell could be operated at temperatures up to 140 °C in a standard EPR cavity. The carbon-based gas diffusion layer showed an EPR signal with a characteristic Dysonian line shape, whose evolution could be monitored in-operando in a non-invasive manner.

  4. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells.

    Niemöller, Arvid; Jakes, Peter; Kayser, Steffen; Lin, Yu; Lehnert, Werner; Granwehr, Josef

    2016-08-01

    Electrochemical cells contain electrically conductive components, which causes various problems if such a cell is analyzed during operation in an EPR resonator. The optimum cell design strongly depends on the application and it is necessary to make certain compromises that need to be individually arranged. Rapid prototyping presents a straightforward option to implement a variable cell design that can be easily adapted to changing requirements. In this communication, it is demonstrated that sample containers produced by 3D printing are suitable for EPR applications, with a particular emphasis on electrochemical applications. The housing of a high temperature polymer electrolyte fuel cell (HT-PEFC) with a phosphoric acid doped polybenzimidazole membrane was prepared from polycarbonate by 3D printing. Using a custom glass Dewar, this fuel cell could be operated at temperatures up to 140°C in a standard EPR cavity. The carbon-based gas diffusion layer showed an EPR signal with a characteristic Dysonian line shape, whose evolution could be monitored in-operando in a non-invasive manner. PMID:27323280

  5. Super-resolved 3-D imaging of live cells organelles from bright-field photon transmission micrographs

    Rychtarikova, Renata; Shi, Kevin; Malakhova, Daria; Machacek, Petr; Smaha, Rebecca; Urban, Jan; Stys, Dalibor

    2016-01-01

    Current biological and medical research is aimed at obtaining a detailed spatiotemporal map of a live cell's interior to describe and predict cell's physiological state. We present here an algorithm for complete 3-D modelling of cellular structures from a z-stack of images obtained using label-free wide-field bright-field light-transmitted microscopy. The method visualizes 3-D objects with a volume equivalent to the area of a camera pixel multiplied by the z-height. The computation is based on finding pixels of unchanged intensities between two consecutive images of an object spread function. These pixels represent strongly light-diffracting, light-absorbing, or light-emitting objects. To accomplish this, variables derived from R\\'{e}nyi entropy are used to suppress camera noise. Using this algorithm, the detection limit of objects is only limited by the technical specifications of the microscope setup--we achieve the detection of objects of the size of one camera pixel. This method allows us to obtain 3-D re...

  6. Electricity Generation with the Novel 3D Electrode from Swim Wastewater in a Dual-chamber Microbial Fuel Cell

    Lai Mei-Feng

    2016-01-01

    Full Text Available The swine wastewater has the characteristics of high concentration of organic matter, suspended solids and more high ammonia nitrogen, odor, complex pollution ingredient and large emissions. Microbial fuel cells (MFC is an electrochemical and biological systems related to chemical energy into electrical energy. A two-chambered cubic microbial fuel cell was used to evaluate the effect of a novel 3D electrode which made of iron and copper on the electricity generation. The swine wastewater containing total chemical oxygen demand (TCOD 3300±300 mg/L was used as the feedstock in anode chamber, and the potassium ferricyanide was used as electron acceptor in cathode chamber. The MFC reactor was incubated with the initial pH 7.0 in a air-shaker with a temperature (ca. 35°C and 100 rpm in fed-batch mode. A fixed external resistance (R of 100 Ω was connected between the electrodes and the closed circuit potentials of the MFCs were recorded every 2 min. The results show that using iron 3D electrode has the peak electricity generation of 176 mV at the first two day and maintained the stable electricity voltage of 110 mV during the 5th to 15th days. The COD removal efficiency could reach 80%. Using copper 3D electrode only can generate the peak electricity of 33.1 mV and stable electricity of 27 mV with the COD removal efficiency of 70%.

  7. Quantitative 3-D imaging of eukaryotic cells using soft x-ray tomography

    Parkinson, Dilworth Y.; McDermott, Gerry; Etkin, Laurence D.; Le Gros, Mark A.; Larabell, Carolyn A.

    2008-01-01

    Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optica...

  8. 3D Imaging of mammalian cells with ion-abrasion scanning electron microscopy

    Heymann, Jurgen A. W.; Shi, Dan; Kim, Sang; Bliss, Donald; Milne, Jacqueline L. S.; Subramaniam, Sriram

    2008-01-01

    Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and mela...

  9. A new 3D tracking method for cell mechanics investigation exploiting the capabilities of digital holography in microscopy

    Miccio, L.; Memmolo, P.; Merola, F.; Fusco, S.; Netti, P. A.; Ferraro, P.

    2014-03-01

    A method for 3D tracking has been developed exploiting Digital Holography features in Microscopy (DHM). In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of samples with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the amplitude refocusing in traditional tracking processes. Moreover, in biology and biomedical research fields one of the main topic is the understanding of morphology and mechanics of cells and microorganisms. Biological samples present low amplitude contrast that limits the information that can be retrieved through optical bright-field microscope measurements. The main effect on light propagating in such objects is in phase. This is known as phase-retardation or phase-shift. DHM is an innovative and alternative approach in microscopy, it's a good candidate for no-invasive and complete specimen analysis because its main characteristic is the possibility to discern between intensity and phase information performing quantitative mapping of the Optical Path Length. In this paper, the flexibility of DH is employed to analyze cell mechanics of unstained cells subjected to appropriate stimuli. DHM is used to measure all the parameters useful to understand the deformations induced by external and controlled stresses on in-vitro cells. Our configuration allows 3D tracking of micro-particles and, simultaneously, furnish quantitative phase-contrast maps. Experimental results are presented and discussed for in vitro cells.

  10. Control of Retinal Ganglion Cell Positioning and Neurite Growth: Combining 3D Printing with Radial Electrospun Scaffolds.

    Kador, Karl E; Grogan, Shawn P; Dorthé, Erik W; Venugopalan, Praseeda; Malek, Monisha F; Goldberg, Jeffrey L; D'lima, Darryl D

    2016-02-01

    Retinal ganglion cells (RGCs) are responsible for the transfer of signals from the retina to the brain. As part of the central nervous system, RGCs are unable to regenerate following injury, and implanted cells have limited capacity to orient and integrate in vivo. During development, secreted guidance molecules along with signals from extracellular matrix and the vasculature guide cell positioning, for example, around the fovea, and axon outgrowth; however, these changes are temporally regulated and are not the same in the adult. Here, we combine electrospun cell transplantation scaffolds capable of RGC neurite guidance with thermal inkjet 3D cell printing techniques capable of precise positioning of RGCs on the scaffold surface. Optimal printing parameters are developed for viability, electrophysiological function and, neurite pathfinding. Different media, commonly used to promote RGC survival and growth, were tested under varying conditions. When printed in growth media containing both brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), RGCs maintained survival and normal electrophysiological function, and displayed radial axon outgrowth when printed onto electrospun scaffolds. These results demonstrate that 3D printing technology may be combined with complex electrospun surfaces in the design of future retinal models or therapies. PMID:26729061

  11. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model

    Mousavi, Seyed Jamaleddin; Hamdy Doweidar, Mohamed

    2015-01-01

    Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs) can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal) together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa), intermediate (20-25 kPa) or hard (30-45 kPa) substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis. PMID:25933372

  12. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model.

    Seyed Jamaleddin Mousavi

    Full Text Available Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa, intermediate (20-25 kPa or hard (30-45 kPa substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis.

  13. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions. PMID:26567131

  14. Impedimetric quantification of cells encapsulated in hydrogel cultured in a paper-based microchamber.

    Lei, Kin Fong; Huang, Chia-Hao; Tsang, Ngan-Ming

    2016-01-15

    Recently, 3D cell culture technique was proposed to provide a more physiologically-meaningful environment for cell-based assays. With the development of microfluidics technology, cellular response can be quantified by impedance measurement technique in a real-time and non-invasive manner. However, handling of these microfluidic systems requires a trained engineering personnel and the operation is not compatible to traditional biological research laboratories. In this work, we incorporated the impedance measurement technique to paper-based 3D cell culture model and demonstrated non-invasive quantification of cells encapsulated in hydrogel during the culture course. A cellulose filter paper was patterned with an array of circular microchambers. Cells were encapsulated in hydrogel and loaded to the microchambers for culturing cells in 3D environment. At the preset schedule during the culture course, the paper was placed on a glass substrate with measurement electrodes for the impedance measurement. Cells in each microchamber was represented by impedance magnitude and cell proliferation could be studied over time. Also, conventional bio-assay was performed to further confirm the feasibility of the impedimetric quantification of cells encapsulated in hydrogel cultured in the paper-based microchamber. This technique provides a convenient, fast, and non-invasive approach to monitor cells cultured in 3D environment. It has potential to be developed for routine 3D cell culture protocol in biological research laboratories. PMID:26592655

  15. 3D Texture Analysis in Renal Cell Carcinoma Tissue Image Grading

    Tae-Yun Kim; Nam-Hoon Cho; Goo-Bo Jeong; Ewert Bengtsson; Heung-Kook Choi

    2014-01-01

    One of the most significant processes in cancer cell and tissue image analysis is the efficient extraction of features for grading purposes. This research applied two types of three-dimensional texture analysis methods to the extraction of feature values from renal cell carcinoma tissue images, and then evaluated the validity of the methods statistically through grade classification. First, we used a confocal laser scanning microscope to obtain image slices of four grades of renal cell carcin...

  16. Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

    Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

    2015-08-01

    In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. PMID:25786567

  17. Enabling Lorentz boosted frame particle-in-cell simulations of laser wakefield acceleration in quasi-3D geometry

    Yu, Peicheng; Xu, Xinlu; Davidson, Asher; Tableman, Adam; Dalichaouch, Thamine; Li, Fei; Meyers, Michael D.; An, Weiming; Tsung, Frank S.; Decyk, Viktor K.; Fiuza, Frederico; Vieira, Jorge; Fonseca, Ricardo A.; Lu, Wei; Silva, Luis O.; Mori, Warren B.

    2016-07-01

    When modeling laser wakefield acceleration (LWFA) using the particle-in-cell (PIC) algorithm in a Lorentz boosted frame, the plasma is drifting relativistically at βb c towards the laser, which can lead to a computational speedup of ∼ γSUB>/bSUB>2 = (1 -space-time distribution of the LWFA data in the lab and boosted frame, we propose to use a moving window to follow the drifting plasma, instead of following the laser driver as is done in the LWFA lab frame simulations, in order to further reduce the computational loads. We describe the details of how the NCI is mitigated for the quasi-3D geometry, the setups for simulations which combine the Lorentz boosted frame, quasi-3D geometry, and the use of a moving window, and compare the results from these simulations against their corresponding lab frame cases. Good agreement is obtained among these sample simulations, particularly when there is no self-trapping, which demonstrates it is possible to combine the Lorentz boosted frame and the quasi-3D algorithms when modeling LWFA. We also discuss the preliminary speedups achieved in these sample simulations.

  18. Fabrication of solution processed 3D nanostructured CuInGaS2 thin film solar cells

    In this study we demonstrate the fabrication of CuInGaS2 (CIGS) thin film solar cells with a three-dimensional (3D) nanostructure based on indium tin oxide (ITO) nanorod films and precursor solutions (Cu, In and Ga nitrates in alcohol). To obtain solution processed 3D nanostructured CIGS thin film solar cells, two different precursor solutions were applied to complete gap filling in ITO nanorods and achieve the desirable absorber film thickness. Specifically, a coating of precursor solution without polymer binder material was first applied to fill the gap between ITO nanorods followed by deposition of the second precursor solution in the presence of a binder to generate an absorber film thickness of ∼1.3 μm. A solar cell device with a (Al, Ni)/AZO/i-ZnO/CdS/CIGS/ITO nanorod/glass structure was constructed using the CIGS film, and the highest power conversion efficiency was measured to be ∼6.3% at standard irradiation conditions, which was 22.5% higher than the planar type of CIGS solar cell on ITO substrate fabricated using the same precursor solutions. (paper)

  19. Organization of Endothelial Cells, Pericytes, and Astrocytes into a 3D Microfluidic in Vitro Model of the Blood-Brain Barrier.

    Wang, Jack D; Khafagy, El-Sayed; Khanafer, Khalil; Takayama, Shuichi; ElSayed, Mohamed E H

    2016-03-01

    The endothelial cells lining the capillaries supplying the brain with oxygen and nutrients form a formidable barrier known as the blood-brain barrier (BBB), which exhibits selective permeability to small drug molecules and virtually impermeable to macromolecular therapeutics. Current in vitro BBB models fail to replicate this restrictive behavior due to poor integration of the endothelial cells with supporting cells (pericytes and astrocytes) following the correct anatomical organization observed in vivo. We report the coculture of mouse brain microvascular endothelial cells (b.End3), pericytes, with/without C8-D1A astrocytes in layered microfluidic channels forming three-dimensional (3D) bi- and triculture models of the BBB. The live/dead assay indicated high viability of all cultured cells up to 21 days. Trans-endothelial electrical resistance (TEER) values confirmed the formation of intact monolayers after 3 days in culture and showed statistically higher values for the triculture model compared to the single and biculture models. Screening the permeability of [(14)C]-mannitol and [(14)C]-urea showed the ability of bi- and triculture models to discriminate between different markers based on their size. Further, permeability of [(14)C]-mannitol across the triculture model after 18 days in culture matched its reported permeability across the BBB in vivo. Mathematical calculations also showed that the radius of the tight junctions pores (R) in the triculture model is similar to the reported diameter of the BBB in vivo. Finally, both the bi- and triculture models exhibited functional expression of the P-glycoprotein efflux pump, which increased with the increase in the number of days in culture. These results collectively indicate that the triculture model is a robust in vitro model of the BBB. PMID:26751280

  20. Usability and Applicability of Microfluidic Cell Culture Systems

    Hemmingsen, Mette

    devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment...... possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs, these...... shown to be needed. This is possibly one of the reasons for the lack of implementation of microfluidic cell culture systems into biological research laboratories. Procedures to perform long-term microfluidic perfusion cell culture experiments have been established. Furthermore, successful application of...

  1. Electric transport in 3D photonic crystal intermediate reflectors for micromorph thin-film tandem solar cells

    Üpping, J.; Bielawny, A.; Lee, S.; Knez, M.; Carius, R.; Wehrspohn, R. B.

    2009-08-01

    The progress of 3D photonic intermediate reflectors for micromorph silicon tandem cells towards a first prototype cell is presented. Intermediate reflectors enhance the absorption of spectrally-selected light in the top cell and decrease the current mismatch between both junctions. A numerical method to predict filter properties for optimal current matching is presented. Our device is an inverted opal structure made of ZnO and fabricated using self-organized nanoparticles and atomic layer deposition for conformal coating. In particular, the influence of ZnO-doping and replicated cracks during drying of the opal is discussed with respect to conductivity and optical properties. A first prototype is compared to a state-of-the-art reference cell.

  2. Osteogenic Differentiation of Miniature Pig Mesenchymal Stem Cells in 2D and 3D Environment

    Juhásová, Jana; Juhás, Štefan; Klíma, Jiří; Strnádel, Ján; Holubová, Monika; Motlík, Jan

    2011-01-01

    Roč. 60, č. 3 (2011), s. 559-571. ISSN 0862-8408 R&D Projects: GA MŠk 1M0538; GA MŠk 2B06130 Institutional research plan: CEZ:AV0Z50450515 Keywords : miniature pig * mesenchymal stem cells * cell differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.555, year: 2011

  3. Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

    Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

    2008-01-01

    Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

  4. Dose distribution and tumor control probability in out-of-field lymph node stations in intensity modulated radiotherapy (IMRT) vs 3D-conformal radiotherapy (3D-CRT) of non-small-cell lung cancer: an in silico analysis

    The advent of IMRT and image-guided radiotherapy (IGRT) in combination with involved-field radiotherapy (IF-RT) in inoperable non-small-cell lung cancer results in a decreased incidental dose deposition in elective nodal stations. While incidental nodal irradiation is considered a relevant by-product of 3D-CRT to control microscopic disease this planning study analyzed the impact of IMRT on dosimetric parameters and tumor control probabilities (TCP) in elective nodal stations in direct comparison with 3D-CRT. The retrospective planning study was performed on 41 patients with NSCLC (stages II-III). The CTV was defined as the primary tumor (GTV + 3 mm) and all FDG-PET-positive lymph node stations. As to the PTV (CTV + 7 mm), both an IMRT plan and a 3D-CRT plan were established. Plans were escalated until the pre-defined dose-constraints of normal tissues (spinal cord, lung, esophagus and heart) were reached. Additionally, IMRT plans were normalized to the total dose of the corresponding 3D-CRT. For two groups of out-of-field mediastinal node stations (all lymph node stations not included in the CTV (LNall-el) and those directly adjacent to the CTV (LNadj-el)) the equivalent uniform dose (EUD) and the TCP (for microscopic disease a D50 of 36.5 Gy was assumed) for the treatment with IMRT vs 3D-CRT were calculated. In comparison, a significantly higher total dose for the PTV could be achieved with the IMRT planning as opposed to conventional 3D-CRT planning (74.3 Gy vs 70.1 Gy; p = 0.03). In identical total reference doses, the EUD of LNadj-el is significantly lower with IMRT than with 3D-CRT (40.4 Gy vs. 44.2 Gy. P = 0.05) and a significant reduction of TCP with IMRT vs 3D-CRT was demonstrated for LNall-el and LNadj-el (12.6 % vs. 14.8 %; and 23.6 % vs 27.3 %, respectively). In comparison with 3D-CRT, IMRT comes along with a decreased EUD in out-of-field lymph node stations. This translates into a statistically significant decrease in TCP-values. Yet, the combination

  5. Topographical guidance of 3D tumor cell migration at an interface of collagen densities

    During cancer progression, metastatic cells leave the primary tumor and invade into the fibrous extracellular matrix (ECM) within the surrounding stroma. This ECM network is highly heterogeneous, and interest in understanding how this network can affect cell behavior has increased in the past several decades. However, replicating this heterogeneity has proven challenging. Here, we designed and utilized a method to create a well-defined interface between two distinct regions of high- and low-density collagen gels to mimic the heterogeneities in density found in the tumor stroma. We show that cells will invade preferentially from the high-density side into the low-density side. We also demonstrate that the net cell migration is a function of the density of the collagen in which the cells are embedded, and the difference in density between the two regions has minimal effect on cell net displacement and distance travelled. Our data further indicate that a low-to-high density interface promotes directional migration and induces formation of focal adhesion on the interface surface. Together, the current results demonstrate how ECM heterogeneities, in the form of interfacial boundaries, can affect cell migration. (paper)

  6. 3D CFD Electrochemical And Heat Transfer Model Of An Internally Manifolded Solid Oxide Electrolysis Cell

    A three-dimensional computational fluid dynamics (CFD) electrochemical model has been created to model high-temperature electrolysis cell performance and steam electrolysis in an internally manifolded planar solid oxide electrolysis cell (SOEC) stack. This design is being evaluated at the Idaho National Laboratory for hydrogen production from nuclear power and process heat. Mass, momentum, energy, and species conservation and transport are provided via the core features of the commercial CFD code FLUENT. A solid-oxide fuel cell (SOFC) model adds the electrochemical reactions and loss mechanisms and computation of the electric field throughout the cell. The FLUENT SOFC user-defined subroutine was modified for this work to allow for operation in the SOEC mode. Model results provide detailed profiles of temperature, operating potential, steam-electrode gas composition, oxygen-electrode gas composition, current density and hydrogen production over a range of stack operating conditions. Single-cell and five-cell results will be presented. Flow distribution through both models is discussed. Flow enters from the bottom, distributes through the inlet plenum, flows across the cells, gathers in the outlet plenum and flows downward making an upside-down ''U'' shaped flow pattern. Flow and concentration variations exist downstream of the inlet holes. Predicted mean outlet hydrogen and steam concentrations vary linearly with current density, as expected. Effects of variations in operating temperature, gas flow rate, oxygen-electrode and steam-electrode current density, and contact resistance from the base case are presented. Contour plots of local electrolyte temperature, current density, and Nernst potential indicate the effects of heat transfer, reaction cooling/heating, and change in local gas composition. Results are discussed for using this design in the electrolysis mode. Discussion of thermal neutral voltage, enthalpy of reaction, hydrogen production, cell thermal

  7. A 3-D Model of Signaling and Transport Pathways in Epithelial Cells

    Quong, A A; Westbrook, C K

    2005-04-01

    A 3-dimensional computer model was developed to simulate the spatial and chemical evolution of calcium ions inside an array of human epithelial kidney cells. This is a prototype model, intended to develop a methodology to incorporate much more complex interactions of metabolic and other processes within many types of cells and lead to increased ability to predict cellular responses to disease as well as to chemical and biological warfare situations. Preliminary tests of the model are described.

  8. HeLa cell response proteome alterations induced by mammalian reovirus T3D infection

    Coombs, Kevin M.

    2013-01-01

    Background Cells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such stud...

  9. Impact of adjustable cryogel properties on the performance of prostate cancer cells in 3D

    Bäcker, A.; Göppert, B.; Sturm, S.; Abaffy, P.; Sollich, T.; Gruhl, F. J.

    2016-01-01

    Background Biochemical and physical characteristics of extracellular environment play a key role in assisting cell behavior over different molecular pathways. In this study, we investigated how the presence of chemical binding sites, the pore network and the stiffness of designed scaffolds affected prostate cancer cells. Methods A blend of poly hydroxyethyl methacrylate–alginate–gelatin scaffold was synthesized by cryogelation process using polyethyleneglycol diacrylate (PEGda) and...

  10. Passivation of ZnO Nanowire Guests and 3D Inverse Opal Host Photoanodes for Dye-Sensitized Solar Cells

    Labouchere, Philippe

    2014-04-23

    A hierarchical host-guest nanostructured photoanode is reported for dye-sensitized solar cells. It is composed of ZnO nanowires grown in situ into the macropores of a 3D ZnO inverse opal structure, which acts both as a seed layer and as a conductive backbone host. Using a combination of self-assembly, hydrothermal or electrodeposition of single crystalline ZnO nanowires and TiO2 passivation, a novel photoanode with scattering capability for optimal light harvesting is fabricated. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Construction of engineering adipose-like tissue in vivo utilizing human insulin gene-modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds.

    Li, Shi-Long; Liu, Yi; Hui, Ling

    2015-12-01

    We evaluated the use of a combination of human insulin gene-modified umbilical cord mesenchymal stromal cells (hUMSCs) with silk fibroin 3D scaffolds for adipose tissue engineering. In this study hUMSCs were isolated and cultured. HUMSCs infected with Ade-insulin-EGFP were seeded in fibroin 3D scaffolds with uniform 50-60 µm pore size. Silk fibroin scaffolds with untransfected hUMSCs were used as control. They were cultured for 4 days in adipogenic medium and transplanted under the dorsal skins of female Wistar rats after the hUMSCs had been labelled with chloromethylbenzamido-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (CM-Dil). Macroscopical impression, fluorescence observation, histology and SEM were used for assessment after transplantation at 8 and 12 weeks. Macroscopically, newly formed adipose tissue was observed in the experimental group and control group after 8 and 12 weeks. Fluorescence observation supported that the formed adipose tissue originated from seeded hUMSCs rather than from possible infiltrating perivascular tissue. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration in the experimental group than in the control group. SEM showed that experimental group cells had more fat-like cells, whose volume was larger than that of the control group, and degradation of the silk fibroin scaffold was greater under SEM observation. This study provides significant evidence that hUMSCs transfected by adenovirus vector have good compatibility with silk fibroin scaffold, and adenoviral transfection of the human insulin gene can be used for the construction of tissue-engineered adipose. PMID:23509085

  12. [Development of three-dimensional breast cancer cell culture drug resistance model].

    Xu, Hong; Liu, Wei; Zhang, Xiu-Zhen; Hou, Liang; Lu, Ying-Jin; Chen, Pei-Pei; Zhang, Can; Feng, Di; Kong, Li; Wang, Xiu-Li

    2016-04-25

    The aim of the present study was to develop three-dimensional (3D) culture model, a more pathologically relevant model, of human breast cancer for drug resistance study. MCF-7 cells were embedded within collagen gel to establish 3D culture model. Cellular morphology was observed using Carmine and HE staining. Cell proliferation was evaluated by CCK-8 assay, and cell activity was detected by Live/Dead staining kit. Drug sensitivities of the 3D culture to doxorubicin, carboplatin, 5-fluorouracil were assayed and compared with those of monolayer (2D) culture. In addition, the levels of drug resistance-related genes P-glycoprotein (P-gp), mrp2 mRNA expressions were detected by real time RT-PCR. Expression level of P-gp protein was detected by Western blot. The results showed that MCF-7 cells in 3D culture formed a number of cell aggregates, and most of them displayed good cell viability. The IC50 values of doxorubicin, carboplatin, 5-fluorouracil were all increased significantly in 3D culture compared with those in 2D culture. Moreover, compared with MCF-7 cells in 2D culture, the cells in 3D culture showed increased mRNA levels of P-gp and mrp2, as well as up-regulated protein expression of P-gp. These results suggest that in vitro collagen-embedded culture system of human breast cancer cells represents an improved pathologically relevant 3D microenvironment for breast cancer cells, providing a robust tool to explore the mechanism of drug resistance of cancer cells. PMID:27108905