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Sample records for 3d cell rearrangements

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

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    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.

  2. Real-time 3D visualization of cellular rearrangements during cardiac valve formation.

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    Pestel, Jenny; Ramadass, Radhan; Gauvrit, Sebastien; Helker, Christian; Herzog, Wiebke; Stainier, Didier Y R

    2016-06-15

    During cardiac valve development, the single-layered endocardial sheet at the atrioventricular canal (AVC) is remodeled into multilayered immature valve leaflets. Most of our knowledge about this process comes from examining fixed samples that do not allow a real-time appreciation of the intricacies of valve formation. Here, we exploit non-invasive in vivo imaging techniques to identify the dynamic cell behaviors that lead to the formation of the immature valve leaflets. We find that in zebrafish, the valve leaflets consist of two sets of endocardial cells at the luminal and abluminal side, which we refer to as luminal cells (LCs) and abluminal cells (ALCs), respectively. By analyzing cellular rearrangements during valve formation, we observed that the LCs and ALCs originate from the atrium and ventricle, respectively. Furthermore, we utilized Wnt/β-catenin and Notch signaling reporter lines to distinguish between the LCs and ALCs, and also found that cardiac contractility and/or blood flow is necessary for the endocardial expression of these signaling reporters. Thus, our 3D analyses of cardiac valve formation in zebrafish provide fundamental insights into the cellular rearrangements underlying this process.

  3. 3D Cell Culture in Alginate Hydrogels

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    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.

  4. 3D culture for cardiac cells.

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    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

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

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    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.

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

    KAUST Repository

    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

  7. "Constructing" the Cell Cycle in 3D

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    Koc, Isil; Turan, Merve

    2012-01-01

    The cycle of duplication and division, known as the "cell cycle," is the essential mechanism by which all living organisms reproduce. This activity allows students to develop an understanding of the main events that occur during the typical eukaryotic cell cycle mostly in the process of mitotic phase that divides the duplicated genetic material…

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

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    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.

  9. An Optically Controlled 3D Cell Culturing System

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    Kelly S. Ishii

    2011-01-01

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

  10. Laser printing of cells into 3D scaffolds

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    Ovsianikov, A; Gruene, M; Koch, L; Maiorana, F; Chichkov, B [Nanotechnology Department, Laser Zentrum Hannover eV, Hollerithallee 8, 30419 Hannover (Germany); Pflaum, M; Wilhelmi, M; Haverich, A, E-mail: a.ovsianikov@lzh.d [Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover (Germany)

    2010-03-15

    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.

  11. 3D printing of biomimetic microstructures for cancer cell migration

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    Huang, Tina Qing; Qu, Xin; Liu, Justin; Chen, Shaochen

    2013-01-01

    To understand the physical behavior and migration of cancer cells, a 3D in vitro micro-chip in hydrogel was created using 3D projection printing. The micro-chip has a honeycomb branched structure, aiming to mimic 3D vascular morphology to test, monitor, and analyze differences in the behavior of cancer cells (i.e. HeLa) vs. non-cancerous cell lines (i.e. 10T1/2). The 3D Projection Printing system can fabricate complex structures in seconds from user-created designs. The fabricated microstructures have three different channel widths of 25, 45, and 120 microns wide to reflect a range of blood vessel diameters. HeLa and 10T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10T1/2 cell migration while HeLa cancer cell migration increased as channel width decreased. This physiologically relevant 3D cancer tissue model has the potential to be a powerful tool for future drug discoveries and cancer migration studies PMID:24150602

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

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    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.

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

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    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.

  14. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

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    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  15. Influence of scaffold design on 3D printed cell constructs.

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    Souness, Auryn; Zamboni, Fernanda; Walker, Gavin M; Collins, Maurice N

    2017-02-14

    Additive manufacturing is currently receiving significant attention in the field of tissue engineering and biomaterial science. The development of precise, affordable 3D printing technologies has provided a new platform for novel research to be undertaken in 3D scaffold design and fabrication. In the past, a number of 3D scaffold designs have been fabricated to investigate the potential of a 3D printed scaffold as a construct which could support cellular life. These studies have shown promising results; however, few studies have utilized a low-cost desktop 3D printing technology as a potential rapid manufacturing route for different scaffold designs. Here six scaffold designs were manufactured using a Fused deposition modeling, a "bottom-up" solid freeform fabrication approach, to determine optimal scaffold architecture for three-dimensional cell growth. The scaffolds, produced from PLA, are coated using pullulan and hyaluronic acid to assess the coating influence on cell proliferation and metabolic rate. Scaffolds are characterized both pre- and postprocessing using water uptake analysis, mechanical testing, and morphological evaluation to study the inter-relationships between the printing process, scaffold design, and scaffold properties. It was found that there were key differences between each scaffold design in terms of porosity, diffusivity, swellability, and compressive strength. An optimal design was chosen based on these physical measurements which were then weighted in accordance to design importance based on literature and utilizing a design matrix technique. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.

  16. 3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells.

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    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.

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

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

    2014-12-15

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

  18. 3D visualization of membrane failures in fuel cells

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    Singh, Yadvinder; Orfino, Francesco P.; Dutta, Monica; Kjeang, Erik

    2017-03-01

    Durability issues in fuel cells, due to chemical and mechanical degradation, are potential impediments in their commercialization. Hydrogen leak development across degraded fuel cell membranes is deemed a lifetime-limiting failure mode and potential safety issue that requires thorough characterization for devising effective mitigation strategies. The scope and depth of failure analysis has, however, been limited by the 2D nature of conventional imaging. In the present work, X-ray computed tomography is introduced as a novel, non-destructive technique for 3D failure analysis. Its capability to acquire true 3D images of membrane damage is demonstrated for the very first time. This approach has enabled unique and in-depth analysis resulting in novel findings regarding the membrane degradation mechanism; these are: significant, exclusive membrane fracture development independent of catalyst layers, localized thinning at crack sites, and demonstration of the critical impact of cracks on fuel cell durability. Evidence of crack initiation within the membrane is demonstrated, and a possible new failure mode different from typical mechanical crack development is identified. X-ray computed tomography is hereby established as a breakthrough approach for comprehensive 3D characterization and reliable failure analysis of fuel cell membranes, and could readily be extended to electrolyzers and flow batteries having similar structure.

  19. 2D- and 3D-culture of cell

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    Khoruzhenko A. I.

    2011-02-01

    Full Text Available The cultivation of mammalian cells in three-dimensional conditions acquires a priority in a variety of biomedical applications. In the areas of toxicology and anticancer drug development it concerns a significant difference of responses to proapoptotic factors of the cells cultured in 2D versus 3D environment. Besides, the clear-cut differences have been found in cell polarity, cytoskeleton structure, distribution of receptors to wide range of hormones, growth factors, etc. in mammalian cells depending on culture conditions. It is resulted in different response of cultured cells to extracellular stimuli. Multicellular spheroids are regarded presently as the most convenient model of solid tumour growth in vitro. The cultivation of thyroid follicles, mammary acini and other structure units, maintaining initial tissue organization, allows studying the behavior, biochemical features and gene profile of differentiated cells. On the other hand, 3D cultures have some limitations in comparison with a well established monolayer culture. The advantages and disadvantages of each type of cultures and their application in biological and medical researches will be discussed in this review

  20. 3D surface topology guides stem cell adhesion and differentiation.

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    Viswanathan, Priyalakshmi; Ondeck, Matthew G; Chirasatitsin, Somyot; Ngamkham, Kamolchanok; Reilly, Gwendolen C; Engler, Adam J; Battaglia, Giuseppe

    2015-06-01

    Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell-substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilizers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors.

  1. 3D-printed external light trap for solar cells.

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    van Dijk, Lourens; Paetzold, Ulrich W; Blab, Gerhard A; Schropp, Ruud E I; di Vece, Marcel

    2016-05-01

    We present a universally applicable 3D-printed external light trap for enhanced absorption in solar cells. The macroscopic external light trap is placed at the sun-facing surface of the solar cell and retro-reflects the light that would otherwise escape. The light trap consists of a reflective parabolic concentrator placed on top of a reflective cage. Upon placement of the light trap, an improvement of 15% of both the photocurrent and the power conversion efficiency in a thin-film nanocrystalline silicon (nc-Si:H) solar cell is measured. The trapped light traverses the solar cell several times within the reflective cage thereby increasing the total absorption in the cell. Consequently, the trap reduces optical losses and enhances the absorption over the entire spectrum. The components of the light trap are 3D printed and made of smoothened, silver-coated thermoplastic. In contrast to conventional light trapping methods, external light trapping leaves the material quality and the electrical properties of the solar cell unaffected. To explain the theoretical operation of the external light trap, we introduce a model that predicts the absorption enhancement in the solar cell by the external light trap. The corresponding calculated path length enhancement shows good agreement with the empirically derived value from the opto-electrical data of the solar cell. Moreover, we analyze the influence of the angle of incidence on the parasitic absorptance to obtain full understanding of the trap performance. © 2015 The Authors. Progress in Photovoltaics: Research and Applications published by John Wiley & Sons, Ltd.

  2. Surface modified alginate microcapsules for 3D cell culture

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    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.

  3. Evolution of chemical bonding and electron density rearrangements during D(3h) → D(3d) reaction in monolayered TiS2: a QTAIM and ELF study.

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    Ryzhikov, Maxim R; Slepkov, Vladimir A; Kozlova, Svetlana G; Gabuda, Svyatoslav P

    2014-08-15

    Monolayered titanium disulfide TiS2, a prospective nanoelectronic material, was previously shown to be subject to an exothermic solid-state D3h -D3d reaction that proceeds via a newly discovered transition state. Here, we study the reaction in detail using topological methods of quantum chemistry (quantum theory of atoms in molecules and electron localization function analysis) and show how electron density and chemical bonding between the atoms change in the course of the reaction. The reaction is shown to undergo a series of topological catastrophes, associated with elementary chemical events such as break and formation of bonds (including the unexpected formation of S-S bonding between sulfur layers), and rearrangement of electron density of outer valence and core shells.

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

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    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.

  5. Rac promotes epithelial cell rearrangement during tracheal tubulogenesis in Drosophila.

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    Chihara, Takahiro; Kato, Kagayaki; Taniguchi, Misako; Ng, Julian; Hayashi, Shigeo

    2003-04-01

    Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. We used the tracheal system of Drosophila to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. We found that a reduced Rac activity led to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibited dynamic epithelial cell rearrangement. Hyperactivation of Rac, on the other hand, inhibited assembly of newly synthesized E-Cadherin into cell junctions and caused loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules.

  6. 3D photospheric velocity field of a Supergranular cell

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    Del Moro, Dario; Berrilli, Francesco

    2007-01-01

    We investigate the plasma flow properties inside a Supergranular (SG) cell, in particular its interaction with small scale magnetic field structures. The SG cell has been identified using the magnetic network (CaII wing brightness) as proxy, applying the TST to high spatial, spectral and temporal resolution observations obtained by IBIS. The full 3D velocity vector field for the SG has been reconstructed at two different photospheric heights. In order to strengthen our findings, we also computed the mean radial flow of the SG by means of cork tracing. We also studied the behaviour of the horizontal and Line of Sight plasma flow cospatial with cluster of bright CaII structures of magnetic origin to better understand the interaction between photospheric convection and small scale magnetic features. The SG cell we investigated seems to be organized with an almost radial flow from its centre to the border. The large scale divergence structure is probably created by a compact region of costant up-flow close to the...

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

    Lifescience Database Archive (English)

    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 ...

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

    Lifescience Database Archive (English)

    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 ...

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

    Lifescience Database Archive (English)

    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 ...

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

    Lifescience Database Archive (English)

    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 ...

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

    Directory of Open Access Journals (Sweden)

    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

  12. Platelet gel: 3D scaffold for cell culture

    OpenAIRE

    Andrei Moroz; Renata Aparecida de Camargo Bittencourt; Sérgio Luis Felisbino; Hamilton da Rosa Pereira; Rosana Rossi-Ferreira; Elenice Deffune

    2009-01-01

    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 PR...

  13. 3D-Printing Crystallographic Unit Cells for Learning Materials Science and Engineering

    Science.gov (United States)

    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…

  14. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    Science.gov (United States)

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

    2016-06-20

    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.

  15. Sliding Hydrogels with Mobile Molecular Ligands and Crosslinks as 3D Stem Cell Niche.

    Science.gov (United States)

    Tong, Xinming; Yang, Fan

    2016-09-01

    The development of a sliding hydrogel with mobile crosslinks and biochemical ligands as a 3D stem cell niche is reported. The molecular mobility of this sliding hydrogel allows stem cells to reorganize the surrounding ligands and change their morphology in 3D. Without changing matrix stiffness, sliding hydrogels support efficient stem cell differentiation toward multiple lineages including adipogenesis, chondrogenesis, and osteogenesis.

  16. A 3D Hydrodynamic Model for Cytokinesis of Eukaryotic Cells

    Science.gov (United States)

    2014-08-01

    division or binary fission, which is the primary method for reproduction. For eukaryotic cells, it’s called cell mitotic process or mitosis . At the late...of cytokinesis in animal, yeast and plant cells. Experimental observations have provide us with a basic picture of cell mitosis . For eu- karyotic... mitosis and cytokinesis in mammalian cells. 2014 Special Focus on Rho GTPases, page e29770, 2014. [7] Ulrike S. Eggert, Timothy J. Mitchison, and

  17. 3D in vitro cell culture models of tube formation

    NARCIS (Netherlands)

    Zegers, M.M.P.

    2014-01-01

    Building the complex architecture of tubular organs is a highly dynamic process that involves cell migration, polarization, shape changes, adhesion to neighboring cells and the extracellular matrix, physicochemical characteristics of the extracellular matrix and reciprocal signaling with the mesench

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

    DEFF Research Database (Denmark)

    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....

  19. Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

    OpenAIRE

    Ahlf Wheatcraft, Dorothy R.; Liu, Xin; Hummon, Amanda B.

    2014-01-01

    Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribu...

  20. New type of cells with multiple chromosome rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Aseeva, Elena A. [National Research Centre for Hematology, Russian Academy of Medical Sciences, Novozykovsky proezd 4a, 125167 Moscow (Russian Federation); Snigiryova, Galina P. [Russian Scientific Centre of Roentgenology and Radiology, ul. Profsoyuznaya 86, 117997 Moscow (Russian Federation); Neverova, Anna L. [National Research Centre for Hematology, Russian Academy of Medical Sciences, Novozykovsky proezd 4a, 125167 Moscow (Russian Federation); Bogomazova, Alexandra N.; Novitskaya, Natalia N.; Khazins, Eva D. [Russian Scientific Centre of Roentgenology and Radiology, ul. Profsoyuznaya 86, 117997 Moscow (Russian Federation); Domracheva, Elena V. [National Research Centre for Hematology, Russian Academy of Medical Sciences, Novozykovsky proezd 4a, 125167 Moscow (Russian Federation)], E-mail: dom@blood.ru

    2010-04-15

    A comparative analysis of the distribution and the frequency of multiaberrant cells (MAC) among lymphocytes in different categories of low dose (up to 0.5 Gy) irradiated people was carried out. The highest MAC frequency was observed in people exposed to {alpha}-radiation (Pu, Rn) and in cosmonauts. This fact allows MAC to be considered as an indicator of a high-energy local exposure. A new type of cells with multiple chromosome rearrangements was discovered in the course of analysis of stable aberrations by the fluorescence in situ hybridization (FISH) method. The biological consequences of MAC formation and possibility of revealing the whole diversity of cells with multiple aberrations by means of modern molecular-cytogenetic methods are discussed.

  1. Bipotent mammary stem cells: now in amazing 3D

    NARCIS (Netherlands)

    van Amerongen, R.

    2014-01-01

    For many decades, developmental biologists and cancer researchers alike have been trying to understand the relationship between the basal and luminal cell compartments in the mouse mammary epithelium. Delineating the mammary stem and progenitor cell hierarchy will provide fundamental knowledge of ho

  2. Biofabrication of cell-loaded 3D spider silk constructs.

    Science.gov (United States)

    Schacht, Kristin; Jüngst, Tomasz; Schweinlin, Matthias; Ewald, Andrea; Groll, Jürgen; Scheibel, Thomas

    2015-02-23

    Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue-like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non-immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell-loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell-binding motif to the spider silk protein further enables fine-tuned control over cell-material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.

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

    OpenAIRE

    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...

  4. Genome rearrangement affects RNA virus adaptability on prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Kendra ePesko

    2015-04-01

    Full Text Available Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wildtype gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense nonsegmented RNA viruses (order Mononegavirales have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV variants with the nucleocapsid (N gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N-gene translocation towards the 5’ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function, especially on PC3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective

  5. Generation of compartmentalized pressure by a nuclear piston governs cell motility in a 3D matrix.

    Science.gov (United States)

    Petrie, Ryan J; Koo, Hyun; Yamada, Kenneth M

    2014-08-29

    Cells use actomyosin contractility to move through three-dimensional (3D) extracellular matrices. Contractility affects the type of protrusions cells use to migrate in 3D, but the mechanisms are unclear. In this work, we found that contractility generated high-pressure lobopodial protrusions in human cells migrating in a 3D matrix. In these cells, the nucleus physically divided the cytoplasm into forward and rear compartments. Actomyosin contractility with the nucleoskeleton-intermediate filament linker protein nesprin-3 pulled the nucleus forward and pressurized the front of the cell. Reducing expression of nesprin-3 decreased and equalized the intracellular pressure. Thus, the nucleus can act as a piston that physically compartmentalizes the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge of the cell to drive lamellipodia-independent 3D cell migration.

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

    Science.gov (United States)

    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.

  7. Local 3D matrix confinement determines division axis through cell shape.

    Science.gov (United States)

    He, Lijuan; Chen, Weitong; Wu, Pei-Hsun; Jimenez, Angela; Wong, Bin Sheng; San, Angela; Konstantopoulos, Konstantinos; Wirtz, Denis

    2016-02-01

    How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via β1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of β1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.

  8. Spontaneous Electroless Galvanic Cell Deposition of 3D Hierarchical and Interlaced S-M-S Heterostructures.

    Science.gov (United States)

    Tan, Chuan Fu; Azmansah, Siti Aishah Bte; Zhu, Hai; Xu, Qing-Hua; Ho, Ghim Wei

    2017-01-01

    One-pot electroless galvanic cell deposition of a 3D hierarchical semiconductor-metal-semiconductor interlaced nanoarray is demonstrated. The fabricated 3D photoanode deviates from the typical planar geometry, and aims to optimize the effective surface area for light harvesting and long-range charge transfer-collection pathways.

  9. 3D tissue formation : the kinetics of human mesenchymal stem cells

    NARCIS (Netherlands)

    Higuera Sierra, Gustavo Andrés

    2010-01-01

    The main thesis in this book proposes that physical phenomena underlies the formation of three-dimensional (3D) tissue. In this thesis, tissue regeneration with mesenchymal stem cells was studied through the law of conservation of mass. MSCs proliferation and 3D tissue formation were explored from 2

  10. Fluid and cell behaviors along a 3D printed alginate/gelatin/fibrin channel.

    Science.gov (United States)

    Xu, Yufan; Wang, Xiaohong

    2015-08-01

    Three-dimensional (3D) cell manipulation is available with the integration of microfluidic technology and rapid prototyping techniques. High-Fidelity (Hi-Fi) constructs hold enormous therapeutic potential for organ manufacturing and regenerative medicine. In the present paper we introduced a quasi-three-dimensional (Q3D) model with parallel biocompatible alginate/gelatin/fibrin hurdles. The behaviors of fluids and cells along the microfluidic channels with various widths were studied. Cells inside the newly designed microfluidic channels attached and grew well. Morphological changes of adipose-derived stem cells (ADSCs) in both two-dimensional (2D) and 3D milieu were found on the printed constructs. Endothelialization occurred with the co-cultures of ADSCs and hepatocytes. This study provides insights into the interactions among fluids, cells and biomaterials, the behaviors of fluids and cells along the microfluidic channels, and the applications of Q3D techniques.

  11. Nonpolarized signaling reveals two distinct modes of 3D cell migration.

    Science.gov (United States)

    Petrie, Ryan J; Gavara, Núria; Chadwick, Richard S; Yamada, Kenneth M

    2012-04-30

    We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.

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

    Science.gov (United States)

    Shen, Chong; Meng, Qin; Zhang, Guoliang

    2014-12-17

    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 (R(2) = 0.92) to the human oral adsorption than that of the Transwell culture (R(2) = 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 3D printing – a key technology for tailored biomedical cell culture lab ware

    Directory of Open Access Journals (Sweden)

    Schmieder Florian

    2016-09-01

    Full Text Available Today’s 3D printing technologies offer great possibilities for biomedical researchers to create their own specific laboratory equipment. With respect to the generation of ex vivo vascular perfusion systems this will enable new types of products that will embed complex 3D structures possibly coupled with cell loaded scaffolds closely reflecting the in-vivo environment. Moreover this could lead to microfluidic devices that should be available in small numbers of pieces at moderate prices. Here, we will present first results of such 3D printed cell culture systems made from plastics and show their use for scaffold based applications.

  18. Examination of 1D Solar Cell Model Limitations Using 3D SPICE Modeling: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    McMahon, W. E.; Olson, J. M.; Geisz, J. F.; Friedman, D. J.

    2012-06-01

    To examine the limitations of one-dimensional (1D) solar cell modeling, 3D SPICE-based modeling is used to examine in detail the validity of the 1D assumptions as a function of sheet resistance for a model cell. The internal voltages and current densities produced by this modeling give additional insight into the differences between the 1D and 3D models.

  19. 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs.

    Science.gov (United States)

    Kolesky, David B; Truby, Ryan L; Gladman, A Sydney; Busbee, Travis A; Homan, Kimberly A; Lewis, Jennifer A

    2014-05-21

    A new bioprinting method is reported for fabricating 3D tissue constructs replete with vasculature, multiple types of cells, and extracellular matrix. These intricate, heterogeneous structures are created by precisely co-printing multiple materials, known as bioinks, in three dimensions. These 3D micro-engineered environments open new -avenues for drug screening and fundamental studies of wound healing, angiogenesis, and stem-cell niches.

  20. Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering.

    Science.gov (United States)

    Lee, Jin Woo; Choi, Yeong-Jin; Yong, Woon-Jae; Pati, Falguni; Shim, Jin-Hyung; Kang, Kyung Shin; Kang, In-Hye; Park, Jaesung; Cho, Dong-Woo

    2016-01-12

    Several studies have focused on the regeneration of liver tissue in a two-dimensional (2D) planar environment, whereas actual liver tissue is three-dimensional (3D). Cell printing technology has been successfully utilized for building 3D structures; however, the poor mechanical properties of cell-laden hydrogels are a major concern. Here, we demonstrate the printing of a 3D cell-laden construct and its application to liver tissue engineering using 3D cell printing technology through a multi-head tissue/organ building system. Polycaprolactone (PCL) was used as a framework material because of its excellent mechanical properties. Collagen bioink containing three different types of cells-hepatocytes (HCs), human umbilical vein endothelial cells , and human lung fibroblasts--was infused into the canals of a PCL framework to induce the formation of capillary--like networks and liver cell growth. A co-cultured 3D microenvironment of the three types of cells was successfully established and maintained. The vascular formation and functional abilities of HCs (i.e., albumin secretion and urea synthesis) demonstrated that the heterotypic interaction among HCs and nonparenchymal cells increased the survivability and functionality of HCs within the collagen gel. Therefore, our results demonstrate the prospect of using cell printing technology for the creation of heterotypic cellular interaction within a structure for liver tissue engineering.

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

    DEFF Research Database (Denmark)

    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......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...

  2. Inhibitors of Rho kinase (ROCK) signaling revert the malignant phenotype of breast cancer cells in 3D context.

    Science.gov (United States)

    Matsubara, Masahiro; Bissell, Mina J

    2016-05-31

    Loss of polarity and quiescence along with increased cellular invasiveness are associated with breast tumor progression. ROCK plays a central role in actin-cytoskeletal rearrangement. We used physiologically relevant 3D cultures of nonmalignant and cancer cells in gels made of laminin-rich extracellular matrix, to investigate ROCK function. Whereas expression levels of ROCK1 and ROCK2 were elevated in cancer cells compared to nonmalignant cells, this was not observed in 2D cultures. Malignant cells showed increased phosphorylation of MLC, corresponding to disorganized F-actin. Inhibition of ROCK signaling restored polarity, decreased disorganization of F-actin, and led to reduction of proliferation. Inhibition of ROCK also decreased EGFR and Integrinβ1 levels, and consequently suppressed activation of Akt, MAPK and FAK as well as GLUT3 and LDHA levels. Again, ROCK inhibition did not inhibit these molecules in 2D. A triple negative breast cancer cell line, which lacks E-cadherin, had high levels of ROCK but was less sensitive to ROCK inhibitors. Exogenous overexpression of E-cadherin, however, rendered these cells strikingly sensitive to ROCK inhibition. Our results add to the growing literature that demonstrate the importance of context and tissue architecture in determining not only regulation of normal and malignant phenotypes but also drug response.

  3. Peptide hydrogels – versatile matrices for 3D cell culture in cancer medicine

    Directory of Open Access Journals (Sweden)

    Peter eWorthington

    2015-04-01

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

  4. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy.

    Science.gov (United States)

    Jiang, Huaidong; Song, Changyong; Chen, Chien-Chun; Xu, Rui; Raines, Kevin S; Fahimian, Benjamin P; Lu, Chien-Hung; Lee, Ting-Kuo; Nakashima, Akio; Urano, Jun; Ishikawa, Tetsuya; Tamanoi, Fuyuhiko; Miao, Jianwei

    2010-06-22

    Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

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

    CERN Document Server

    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.

  6. Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels

    Science.gov (United States)

    Lai, Janice H.; Kajiyama, Glen; Smith, Robert Lane; Maloney, William; Yang, Fan

    2013-12-01

    Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

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

    Science.gov (United States)

    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.

  8. 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

    Science.gov (United States)

    Bédouin, Y; Pellen Mussi, P; Tricot-Doleux, S; Chauvel-Lebret, D; Auroy, P; Ravalec, X; Oudadesse, H; Perez, F

    2015-01-01

    This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chih-Hao [Department of Electrical Engineering, I-Shou University, Taiwan, ROC (China); Neurosurgery, Department of Surgery, Kaohsiung Veterans General Hospital, Taiwan, ROC (China); Department of Biomedical Engineering, I-Shou University, Taiwan, ROC (China); Kuo, Shyh Ming [Department of Biomedical Engineering, I-Shou University, Taiwan, ROC (China); Liu, Guei-Sheung [Centre for Eye Research Australia, University of Melbourne (Australia); Chen, Wan-Nan U. [Department of Biological Science and Technology, I-Shou University, Taiwan, ROC (China); Chuang, Chin-Wen [Department of Electrical Engineering, I-Shou University, Taiwan, ROC (China); Liu, Li-Feng, E-mail: liulf@isu.edu.tw [Department of Biological Science and Technology, I-Shou University, Taiwan, ROC (China)

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. Black-Right-Pointing-Pointer Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. Black-Right-Pointing-Pointer 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 {mu}m 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.

  10. Hypoxia Created Human Mesenchymal Stem Cell Sheet for Prevascularized 3D Tissue Construction.

    Science.gov (United States)

    Zhang, Lijun; Xing, Qi; Qian, Zichen; Tahtinen, Mitchell; Zhang, Zhaoqiang; Shearier, Emily; Qi, Shaohai; Zhao, Feng

    2016-02-01

    3D tissue based on human mesenchymal stem cell (hMSC) sheets offers many interesting opportunities for regenerating multiple types of connective tissues. Prevascularizing hMSC sheets with endothelial cells (ECs) will improve 3D tissue performance by supporting cell survival and accelerating integration with host tissue. It is hypothesized that hypoxia cultured hMSC sheets can promote microvessel network formation and preserve stemness of hMSCs. This study investigates the vascularization of hMSC sheets under different oxygen tensions. It is found that the HN condition, in which hMSC sheets formed under physiological hypoxia (2% O2 ) and then cocultured with ECs under normoxia (20% O2 ), enables longer and more branched microvessel network formation. The observation is corroborated by higher levels of angiogenic factors in coculture medium. Additionally, the hypoxic hMSC sheet is more uniform and less defective, which facilitates fabrication of 3D prevascularized tissue construct by layering the prevascularized hMSC sheets and maturing in rotating wall vessel bioreactor. The hMSCs in the 3D construct still maintain multilineage differentiation ability, which indicates the possible application of the 3D construct for various connective tissues regeneration. These results demonstrate that hypoxia created hMSC sheets benefit the microvessel growth and it is feasible to construct 3D prevascularized tissue construct using the prevascularized hMSC sheets.

  11. 3D texture analysis in renal cell carcinoma tissue image grading.

    Science.gov (United States)

    Kim, Tae-Yun; Cho, Nam-Hoon; Jeong, Goo-Bo; Bengtsson, Ewert; Choi, Heung-Kook

    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 carcinoma, which were then reconstructed into 3D volumes. Next, we extracted quantitative values using a 3D gray level cooccurrence matrix (GLCM) and a 3D wavelet based on two types of basis functions. To evaluate their validity, we predefined 6 different statistical classifiers and applied these to the extracted feature sets. In the grade classification results, 3D Haar wavelet texture features combined with principal component analysis showed the best discrimination results. Classification using 3D wavelet texture features was significantly better than 3D GLCM, suggesting that the former has potential for use in a computer-based grading system.

  12. 3D Texture Analysis in Renal Cell Carcinoma Tissue Image Grading

    Directory of Open Access Journals (Sweden)

    Tae-Yun Kim

    2014-01-01

    Full Text Available 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 carcinoma, which were then reconstructed into 3D volumes. Next, we extracted quantitative values using a 3D gray level cooccurrence matrix (GLCM and a 3D wavelet based on two types of basis functions. To evaluate their validity, we predefined 6 different statistical classifiers and applied these to the extracted feature sets. In the grade classification results, 3D Haar wavelet texture features combined with principal component analysis showed the best discrimination results. Classification using 3D wavelet texture features was significantly better than 3D GLCM, suggesting that the former has potential for use in a computer-based grading system.

  13. 3D Cell Printing of Functional Skeletal Muscle Constructs Using Skeletal Muscle-Derived Bioink.

    Science.gov (United States)

    Choi, Yeong-Jin; Kim, Taek Gyoung; Jeong, Jonghyeon; Yi, Hee-Gyeong; Park, Ji Won; Hwang, Woonbong; Cho, Dong-Woo

    2016-10-01

    Engineered skeletal muscle tissues that mimic the structure and function of native muscle have been considered as an alternative strategy for the treatment of various muscular diseases and injuries. Here, it is demonstrated that 3D cell-printing of decellularized skeletal muscle extracellular matrix (mdECM)-based bioink facilitates the fabrication of functional skeletal muscle constructs. The cellular alignment and the shape of the tissue constructs are controlled by 3D cell-printing technology. mdECM bioink provides the 3D cell-printed muscle constructs with a myogenic environment that supports high viability and contractility as well as myotube formation, differentiation, and maturation. More interestingly, the preservation of agrin is confirmed in the mdECM, and significant increases in the formation of acetylcholine receptor clusters are exhibited in the 3D cell-printed muscle constructs. In conclusion, mdECM bioink and 3D cell-printing technology facilitate the mimicking of both the structural and functional properties of native muscle and hold great promise for producing clinically relevant engineered muscle for the treatment of muscular injuries.

  14. Quantification by SIFT-MS of acetaldehyde released by lung cells in a 3D model.

    Science.gov (United States)

    Rutter, Abigail V; Chippendale, Thomas W E; Yang, Ying; Španěl, Patrik; Smith, David; Sulé-Suso, Josep

    2013-01-07

    Our previous studies have shown that both lung cancer cells and non-malignant lung cells release acetaldehyde in vitro. However, data from other laboratories have produced conflicting results. Furthermore, all these studies have been carried out in 2D models which are less physiological cell growth systems when compared to 3D models. Therefore, we have carried out further work on the release of acetaldehyde by lung cells in 3D collagen hydrogels. Lung cancer cells CALU-1 and non-malignant lung cells NL20 were seeded in these hydrogels at different cell concentrations and the release of acetaldehyde was measured with the Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) technique. The data obtained showed that the amount of acetaldehyde released by both cell types grown in a 3D model is higher when compared to that of the same cells grown in 2D models. More importantly, acetaldehyde from the headspace of lung cancer cells could be measured even at a low cell concentration (10(5) cells per hydrogel). The differential of acetaldehyde release could be, depending on the cell concentration, more than 3 fold higher for cancer cells when compared to non-malignant lung cells. This pilot study is the first to study acetaldehyde emission from albeit only two cell types cultured in 3D scaffolds. Clearly, from such limited data the behaviour of other cell types and of tumour cells in vivo cannot be predicted with confidence. Nevertheless, this work represents another step in the search for volatile biomarkers of tumour cells, the ultimate goal of which is to exploit volatile compounds in exhaled breath and other biological fluids as biomarkers of tumours in vivo.

  15. 3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

    Science.gov (United States)

    Chang, Lingqian

    Electroporation is one of the most common non-viral methods for gene delivery. Recent progress in gene therapy has offered special opportunities to electroporation for in vitro and in vivo applications. However, conventional bulk electroporation (BEP) inevitably causes serious cell damage and stochastic transfection between cells. Microfluidic electroporation (MEP) has been claimed to provide benign single cell transfection for the last decade. Nevertheless, the intracellular transport in both MEP and BEP systems is highly diffusion-dominant, which prevents precise dose control and high uniformity. In this Ph.D. research, we developed a 3D nanochannel-electroporation (3D NEP) platform for mass cell transfection. A silicon-based nanochannel array (3D NEP) chip was designed and fabricated for cell manipulation and electroporation. The chip, designed as Z-directional microchannel - nanochannel array, was fabricated by clean room techniques including projection photolithography and deep reactive-ion etching (DRIE). The fabricated 3D NEP chip is capable of handling 40,000 cells per 1 cm2, up to 1 million per wafer (100 mm diameter). High-throughput cell manipulation technologies were investigated for precise alignment of individual cells to the nanochannel array, a key step for NEP to achieve dose control. We developed three techniques for cell trapping in this work. (1) Magnetic tweezers (MTs) were integrated on the chip to remotely control cells under a programmed magnetic field. (2) A positive dielectrophoresis (pDEP) power system was built as an alternative to trap cells onto the nanochannel array using DEP force. (3) A novel yet simple 'dipping-trap' method was used to rapidly trap cells onto a nanochannel array, aligned by a micro-cap array pattern on the 3D NEP chip, which eventually offered 70 - 90 % trapping efficiency and 90 % specificity. 3D NEP platforms were assembled for cell transfection based on the Si-based nanochannel array chip and cell manipulation

  16. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Machado, C B [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Ventura, J M G [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Lemos, A F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Ferreira, J M F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Leite, M F [Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Goes, A M [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil)

    2007-06-01

    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.

  17. Collective dynamics of cell migration and cell rearrangements

    Science.gov (United States)

    Kabla, Alexandre

    Understanding multicellular processes such as embryo development or cancer metastasis requires to decipher the contributions of local cell autonomous behaviours and long range interactions with the tissue environment. A key question in this context concerns the emergence of large scale coordination in cell behaviours, a requirement for collective cell migration or convergent extension. I will present a few examples where physical and mechanical aspects play a significant role in driving tissue scale dynamics. Geometrical confinement is one of the key external factors influencing large scale coordination during collective migration. Using a combination of in vitro experiments and numerical simulations, we show that the velocity correlation length, measured in unconfined conditions, provides a convenient length scale to predict the dynamic response under confinement. The same length scale can also be used to quantify the influence range of directional cues within the cell population. Heterogeneity within motile cell populations is frequently associated with an increase in their invasive capability and appears to play an important role during cancer metastasis. Using in silico experiments, we studied the way cell invasion is influenced by both the degree of cell coordination and the amount of variability in the motile force of the invading cells. Results suggest that mechanical heterogeneity dramatically enhances the invasion rate through an emerging cooperative process between the stronger and weaker cells, accounting for a number of observed invasion phenotypes. Effective convergent extension requires on a consistent orientation of cell intercalation at the tissue scale, most often in relation with planar cell polarity mechanisms to define the primary axes of deformation. Using a novel modelling approach for cells mechanical interactions, we studied the dynamics of substrate free motile cell populations. Ongoing work shows in particular that nematic order emerges

  18. 3D-printed concentrator arrays for external light trapping on thin film solar cells

    NARCIS (Netherlands)

    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

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

    OpenAIRE

    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...

  20. 3D reconstitution of the patterned neural tube from embryonic stem cells.

    Science.gov (United States)

    Meinhardt, Andrea; Eberle, Dominic; Tazaki, Akira; Ranga, Adrian; Niesche, Marco; Wilsch-Bräuninger, Michaela; Stec, Agnieszka; Schackert, Gabriele; Lutolf, Matthias; Tanaka, Elly M

    2014-12-09

    Inducing organogenesis in 3D culture is an important aspect of stem cell research. Anterior neural structures have been produced from large embryonic stem cell (ESC) aggregates, but the steps involved in patterning such complex structures have been ill defined, as embryoid bodies typically contained many cell types. Here we show that single mouse ESCs directly embedded in Matrigel or defined synthetic matrices under neural induction conditions can clonally form neuroepithelial cysts containing a single lumen in 3D. Untreated cysts were uniformly dorsal and could be ventralized to floor plate (FP). Retinoic acid posteriorized cysts to cervical levels and induced localize FP formation yielding full patterning along the dorsal/ventral (DV) axis. Correct spatial organization of motor neurons, interneurons, and dorsal interneurons along the DV axis was observed. This system serves as a valuable tool for studying morphogen action in 3D and as a source of patterned spinal cord tissue.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

    Science.gov (United States)

    Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

    2013-08-09

    : The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

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

    Directory of Open Access Journals (Sweden)

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

    Science.gov (United States)

    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.

  5. Modeling spatial distribution of oxygen in 3d culture of islet beta-cells.

    Science.gov (United States)

    McReynolds, John; Wen, Yu; Li, Xiaofei; Guan, Jianjun; Jin, Sha

    2017-01-01

    Three-dimensional (3D) scaffold culture of pancreatic β-cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β-cells is that β-cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β-cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell-laden collagen culture on β-cell proliferation, a culture model with encapsulation of an oxygen-generator was established. The oxygen-generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the β-cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial-temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation-aided 3D culture would augment the oxygen supply required for the β-cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell-laden scaffold cultures with an in situ oxygen supply significantly improved the β-cells' biological function. These β-cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional β-cell cultures. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221-228, 2017.

  6. Bioengineered 3D Glial Cell Culture Systems and Applications for Neurodegeneration and Neuroinflammation.

    Science.gov (United States)

    Watson, P Marc D; Kavanagh, Edel; Allenby, Gary; Vassey, Matthew

    2017-02-01

    Neurodegeneration and neuroinflammation are key features in a range of chronic central nervous system (CNS) diseases such as Alzheimer's and Parkinson's disease, as well as acute conditions like stroke and traumatic brain injury, for which there remains significant unmet clinical need. It is now well recognized that current cell culture methodologies are limited in their ability to recapitulate the cellular environment that is present in vivo, and there is a growing body of evidence to show that three-dimensional (3D) culture systems represent a more physiologically accurate model than traditional two-dimensional (2D) cultures. Given the complexity of the environment from which cells originate, and their various cell-cell and cell-matrix interactions, it is important to develop models that can be controlled and reproducible for drug discovery. 3D cell models have now been developed for almost all CNS cell types, including neurons, astrocytes, microglia, and oligodendrocyte cells. This review will highlight a number of current and emerging techniques for the culture of astrocytes and microglia, glial cell types with a critical role in neurodegenerative and neuroinflammatory conditions. We describe recent advances in glial cell culture using electrospun polymers and hydrogel macromolecules, and highlight how these novel culture environments influence astrocyte and microglial phenotypes in vitro, as compared to traditional 2D systems. These models will be explored to illuminate current trends in the techniques used to create 3D environments for application in research and drug discovery focused on astrocytes and microglial cells.

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

    Directory of Open Access Journals (Sweden)

    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. New data-driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume.

    Science.gov (United States)

    Roselli, L; Paparella, F; Stanca, E; Basset, A

    2015-06-01

    Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex-shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex-shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data-driven formula-based approach to the calculation of biovolume of this complex-shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.

  9. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

    Science.gov (United States)

    Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

    2015-01-01

    3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.

  10. 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways.

    Science.gov (United States)

    DelNero, Peter; Lane, Maureen; Verbridge, Scott S; Kwee, Brian; Kermani, Pouneh; Hempstead, Barbara; Stroock, Abraham; Fischbach, Claudia

    2015-07-01

    Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation.

  11. Primed 3D injectable microniches enabling low-dosage cell therapy for critical limb ischemia.

    Science.gov (United States)

    Li, Yaqian; Liu, Wei; Liu, Fei; Zeng, Yang; Zuo, Simin; Feng, Siyu; Qi, Chunxiao; Wang, Bingjie; Yan, Xiaojun; Khademhosseini, Ali; Bai, Jing; Du, Yanan

    2014-09-16

    The promise of cell therapy for repair and restoration of damaged tissues or organs relies on administration of large dose of cells whose healing benefits are still limited and sometimes irreproducible due to uncontrollable cell loss and death at lesion sites. Using a large amount of therapeutic cells increases the costs for cell processing and the risks of side effects. Optimal cell delivery strategies are therefore in urgent need to enhance the specificity, efficacy, and reproducibility of cell therapy leading to minimized cell dosage and side effects. Here, we addressed this unmet need by developing injectable 3D microscale cellular niches (microniches) based on biodegradable gelatin microcryogels (GMs). The microniches are constituted by in vitro priming human adipose-derived mesenchymal stem cells (hMSCs) seeded within GMs resulting in tissue-like ensembles with enriched extracellular matrices and enhanced cell-cell interactions. The primed 3D microniches facilitated cell protection from mechanical insults during injection and in vivo cell retention, survival, and ultimate therapeutic functions in treatment of critical limb ischemia (CLI) in mouse models compared with free cell-based therapy. In particular, 3D microniche-based therapy with 10(5) hMSCs realized better ischemic limb salvage than treatment with 10(6) free-injected hMSCs, the minimum dosage with therapeutic effects for treating CLI in literature. To the best of our knowledge, this is the first convincing demonstration of injectable and primed cell delivery strategy realizing superior therapeutic efficacy for treating CLI with the lowest cell dosage in mouse models. This study offers a widely applicable cell delivery platform technology to boost the healing power of cell regenerative therapy.

  12. Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix.

    Science.gov (United States)

    Panorchan, Porntula; Lee, Jerry S H; Kole, Thomas P; Tseng, Yiider; Wirtz, Denis

    2006-11-01

    Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

  13. Fabrication of 3D cell-laden hydrogel microstructures through photo-mold patterning.

    Science.gov (United States)

    Occhetta, P; Sadr, N; Piraino, F; Redaelli, A; Moretti, M; Rasponi, M

    2013-09-01

    Native tissues are characterized by spatially organized three-dimensional (3D) microscaled units which functionally define cells-cells and cells-extracellular matrix interactions. The ability to engineer biomimetic constructs mimicking these 3D microarchitectures is subject to the control over cell distribution and organization. In the present study we introduce a novel protocol to generate 3D cell laden hydrogel micropatterns with defined size and shape. The method, named photo-mold patterning (PMP), combines hydrogel micromolding within polydimethylsiloxane (PDMS) stamps and photopolymerization through a recently introduced biocompatible ultraviolet (UVA) activated photoinitiator (VA-086). Exploiting PDMS micromolds as geometrical constraints for two methacrylated prepolymers (polyethylene glycol diacrylate and gelatin methacrylate), micrometrically resolved structures were obtained within a 3 min exposure to a low cost and commercially available UVA LED. The PMP was validated both on a continuous cell line (human umbilical vein endothelial cells expressing green fluorescent protein, HUVEC GFP) and on primary human bone marrow stromal cells (BMSCs). HUVEC GFP and BMSCs were exposed to 1.5% w/v VA-086 and UVA light (1 W, 385 nm, distance from sample = 5 cm). Photocrosslinking conditions applied during the PMP did not negatively affect cells viability or specific metabolic activity. Quantitative analyses demonstrated the potentiality of PMP to uniformly embed viable cells within 3D microgels, creating biocompatible and favorable environments for cell proliferation and spreading during a seven days' culture. PMP can thus be considered as a promising and cost effective tool for designing spatially accurate in vitro models and, in perspective, functional constructs.

  14. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods.

    Science.gov (United States)

    Gildea, John J; McGrath, Helen E; Van Sciver, Robert E; Wang, Dora Bigler; Felder, Robin A

    2013-01-01

    The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

  15. In Vivo Chondrogenesis in 3D Bioprinted Human Cell-laden Hydrogel Constructs

    Science.gov (United States)

    Möller, Thomas; Hägg, Daniel; Brantsing, Camilla; Rotter, Nicole; Apelgren, Peter; Lindahl, Anders; Kölby, Lars; Gatenholm, Paul

    2017-01-01

    Background: The three-dimensional (3D) bioprinting technology allows creation of 3D constructs in a layer-by-layer fashion utilizing biologically relevant materials such as biopolymers and cells. The aim of this study is to investigate the use of 3D bioprinting in a clinically relevant setting to evaluate the potential of this technique for in vivo chondrogenesis. Methods: Thirty-six nude mice (Balb-C, female) received a 5- × 5- × 1-mm piece of bioprinted cell-laden nanofibrillated cellulose/alginate construct in a subcutaneous pocket. Four groups of printed constructs were used: (1) human (male) nasal chondrocytes (hNCs), (2) human (female) bone marrow–derived mesenchymal stem cells (hBMSCs), (3) coculture of hNCs and hBMSCs in a 20/80 ratio, and (4) Cell-free scaffolds (blank). After 14, 30, and 60 days, the scaffolds were harvested for histological, immunohistochemical, and mechanical analysis. Results: The constructs had good mechanical properties and keep their structural integrity after 60 days of implantation. For both the hNC constructs and the cocultured constructs, a gradual increase of glycosaminoglycan production and hNC proliferation was observed. However, the cocultured group showed a more pronounced cell proliferation and enhanced deposition of human collagen II demonstrated by immunohistochemical analysis. Conclusions: In vivo chondrogenesis in a 3D bioprinted human cell-laden hydrogel construct has been demonstrated. The trophic role of the hBMSCs in stimulating hNC proliferation and matrix deposition in the coculture group suggests the potential of 3D bioprinting of human cartilage for future application in reconstructive surgery. PMID:28280669

  16. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

    NARCIS (Netherlands)

    Ricci, C.; Mota, C.M.; Moscato, S.; Alessandro, D' D.; Ugel, S.; Sartoris, S.; Bronte, V.; Boggi, U.; Campani, D.; Funel, N.; Moroni, L.; Danti, S.

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol

  17. Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

    Directory of Open Access Journals (Sweden)

    Ken Ye

    Full Text Available Infrapatellar fat pad adipose stem cells (IPFP-ASCs have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a 'cap' on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

  18. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    Science.gov (United States)

    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.

  19. 3D culture increases pluripotent gene expression in mesenchymal stem cells through relaxation of cytoskeleton tension.

    Science.gov (United States)

    Zhou, Ying; Chen, Haiyan; Li, Hong; Wu, Yaojiong

    2017-03-09

    Three-dimensional (3D) culture has been shown to improve pluripotent gene expression in mesenchymal stem cells (MSCs), but the underlining mechanisms were poorly understood. Here, we found that the relaxation of cytoskeleton tension of MSCs in 3D culture was critically associated with the expressional up-regulation of Nanog. Cultured in spheroids, MSCs showed decreased integrin-based cell-matrix adhesion but increased cadherin-based cell-cell interaction. Different from that in 2D culture, where MSCs exhibited branched and multiple-directed F-actin stress bundles at the cell edge and strengthened stress fibres transversing the cell body, MSCs cultured in spheroids showed compact cell body, relaxed cytoskeleton tension with very thin cortical actin filament outlining the cell, and increased expression of Nanog along with reduced levels of Suv39h1 (H3K9 methyltransferase) and H3K9me3. Notably, pharmaceutical inhibition of actin polymerization with cytochalasin D or silencing Suv39h1 expression with siRNA in 2D-cultured MSCs elevated the expression of Nanog via H3K9 demethylation. Thus, our data suggest that 3D culture increases the expression of Nanog through the relaxation of actin cytoskeleton, which mediates reduced Suv39h1 and H3K9me3 levels.

  20. Rapid Fabrication of Cell-Laden Alginate Hydrogel 3D Structures by Micro Dip-Coating

    Science.gov (United States)

    Ghanizadeh Tabriz, Atabak; Mills, Christopher G.; Mullins, John J.; Davies, Jamie A.; Shu, Wenmiao

    2017-01-01

    Development of a simple, straightforward 3D fabrication method to culture cells in 3D, without relying on any complex fabrication methods, remains a challenge. In this paper, we describe a new technique that allows fabrication of scalable 3D cell-laden hydrogel structures easily, without complex machinery: the technique can be done using only apparatus already available in a typical cell biology laboratory. The fabrication method involves micro dip-coating of cell-laden hydrogels covering the surface of a metal bar, into the cross-linking reagents calcium chloride or barium chloride to form hollow tubular structures. This method can be used to form single layers with thickness ranging from 126 to 220 µm or multilayered tubular structures. This fabrication method uses alginate hydrogel as the primary biomaterial and a secondary biomaterial can be added depending on the desired application. We demonstrate the feasibility of this method, with survival rate over 75% immediately after fabrication and normal responsiveness of cells within these tubular structures using mouse dermal embryonic fibroblast cells and human embryonic kidney 293 cells containing a tetracycline-responsive, red fluorescent protein (tHEK cells). PMID:28286747

  1. Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda.

    Directory of Open Access Journals (Sweden)

    Laura E Valverde-Islas

    Full Text Available BACKGROUND: Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function. METHODOLOGY/PRINCIPAL FINDINGS: Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells. CONCLUSIONS/SIGNIFICANCE: We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.

  2. Stochastic microstructure modeling and electrochemical simulation of lithium-ion cell anodes in 3D

    Science.gov (United States)

    Hein, Simon; Feinauer, Julian; Westhoff, Daniel; Manke, Ingo; Schmidt, Volker; Latz, Arnulf

    2016-12-01

    Thermodynamically consistent transport theory is used to compare 3D images of real anode microstructures from lithium-ion batteries to virtual ones created by a parametric stochastic 3D microstructure model. Half-cell simulations in 3D with spatially resolved microstructures at different applied currents show that for low currents the deviations between various electrochemical quantities like current density or overpotential are negligibly small. For larger currents small differences become more pronounced. Qualitative and quantitative differences of these features are discussed with respect to the microstructure and it is shown that the real and virtual structures behave similar during electrochemical simulations. Extensions of the stochastic microstructure model, which overcome small differences in electrochemical behavior, are proposed.

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

    DEFF Research Database (Denmark)

    Jørgensen, Peter Stanley

    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...... task of manually delineating structures within each image slice or the quality of manual and automatic segmentation schemes. To solve this, a framework for the automatic segmentation of 3D image data is developed. The technique is based on a level set method and uses numerical approximations to partial...... differential equations to evolve a 3D surface to capture the phase boundaries. Vector fields derived from the experimentally acquired data are used as the driving forces. The framework performs the segmentation in 3D rather than on a slice by slice basis. It naturally supplies sub-voxel accuracy of segmented...

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Investigation of osteoblast cells behavior in polymeric 3D micropatterned scaffolds using digital holographic microscopy.

    Science.gov (United States)

    Mihailescu, M; Popescu, R C; Matei, A; Acasandrei, A; Paun, I A; Dinescu, M

    2014-08-01

    The effect of micropatterned polymeric scaffolds on the features of the cultured cells at different time intervals after seeding was investigated by digital holographic microscopy. Both parallel and perpendicular walls, with different heights, were fabricated using two-photon lithography on photopolymers. The walls were subsequently coated with polypyrrole-based thin films using the matrix assisted pulsed laser evaporation technique. Osteoblast-like cells, MG-63 line, were cultured on these polymeric 3D micropatterned scaffolds. To analyze these scaffolds with/without cultured cells, an inverted digital holographic microscope, which provides 3D images, was used. Information about the samples' refractive indices and heights was obtained from the phase shift introduced in the optical path. Characteristics of cell adhesion, alignment, orientation, and morphology as a function of the wall heights and time from seeding were highlighted.

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

    Science.gov (United States)

    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.

  7. Thermoforming techniques for manufacturing porous scaffolds for application in 3D cell cultivation.

    Science.gov (United States)

    Borowiec, Justyna; Hampl, Jörg; Gebinoga, Michael; Elsarnagawy, Tarek; Elnakady, Yasser A; Fouad, Hassan; Almajhadi, Fahd; Fernekorn, Uta; Weise, Frank; Singh, Sukhdeep; Elsarnagawy, Dief; Schober, Andreas

    2015-04-01

    Within the scientific community, there is an increasing demand to apply advanced cell cultivation substrates with increased physiological functionalities for studying spatially defined cellular interactions. Porous polymeric scaffolds are utilized for mimicking an organ-like structure or engineering complex tissues and have become a key element for three-dimensional (3D) cell cultivation in the meantime. As a consequence, efficient 3D scaffold fabrication methods play an important role in modern biotechnology. Here, we present a novel thermoforming procedure for manufacturing porous 3D scaffolds from permeable materials. We address the issue of precise thermoforming of porous polymer foils by using multilayer polymer thermoforming technology. This technology offers a new method for structuring porous polymer foils that are otherwise available for non-porous polymers only. We successfully manufactured 3D scaffolds from solvent casted and phase separated polylactic acid (PLA) foils and investigated their biocompatibility and basic cellular performance. The HepG2 cell culture in PLA scaffold has shown enhanced albumin secretion rate in comparison to a previously reported polycarbonate based scaffold with similar geometry.

  8. 3D imaging of Sox2 enhancer clusters in embryonic stem cells.

    Science.gov (United States)

    Liu, Zhe; Legant, Wesley R; Chen, Bi-Chang; Li, Li; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert

    2014-12-24

    Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

  9. Polymer-based mesh as supports for multi-layered 3D cell culture and assays.

    Science.gov (United States)

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

    2014-01-01

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

  10. A 3D in vitro bone organ model using human progenitor cells

    Directory of Open Access Journals (Sweden)

    A Papadimitropoulos

    2011-05-01

    Full Text Available Three-dimensional (3D organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0 ±0.3%, pre/osteoclastic (14.0 ±1.4% and mesenchymal/osteoblastic (44.0 ±8.4% phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+ osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides, resorption (by N-terminus collagen-I telopeptides and phosphate levels and osteoclastic activity (by TRAP-5b only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants.

  11. Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments.

    Science.gov (United States)

    Greiner, Alexandra M; Klein, Franziska; Gudzenko, Tetyana; Richter, Benjamin; Striebel, Thomas; Wundari, Bayu G; Autenrieth, Tatjana J; Wegener, Martin; Franz, Clemens M; Bastmeyer, Martin

    2015-11-01

    Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, β1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

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

    Science.gov (United States)

    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.

  13. 3D cell culture: a review of current approaches and techniques.

    Science.gov (United States)

    Haycock, John W

    2011-01-01

    Cell culture in two dimensions has been routinely and diligently undertaken in thousands of laboratories worldwide for the past four decades. However, the culture of cells in two dimensions is arguably primitive and does not reproduce the anatomy or physiology of a tissue for informative or useful study. Creating a third dimension for cell culture is clearly more relevant, but requires a multidisciplinary approach and multidisciplinary expertise. When entering the third dimension, investigators need to consider the design of scaffolds for supporting the organisation of cells or the use of bioreactors for controlling nutrient and waste product exchange. As 3D culture systems become more mature and relevant to human and animal physiology, the ability to design and develop co-cultures becomes possible as does the ability to integrate stem cells. The primary objectives for developing 3D cell culture systems vary widely - and range from engineering tissues for clinical delivery through to the development of models for drug screening. The intention of this review is to provide a general overview of the common approaches and techniques for designing 3D culture models.

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

    Science.gov (United States)

    Zonca, Michael R., Jr.

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

  15. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Science.gov (United States)

    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

  16. Rab3D is critical for secretory granule maturation in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Tanja Kögel

    Full Text Available Neuropeptide- and hormone-containing secretory granules (SGs are synthesized at the trans-Golgi network (TGN as immature secretory granules (ISGs and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs. Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

  17. A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

    Science.gov (United States)

    Wiley, Luke A; Beebe, David C; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-05-12

    This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

  18. Fractal dimension as a measure of altered actin cytoskeleton in MC3T3-E1 cells under simulated microgravity using 3-D/2-D clinostats.

    Science.gov (United States)

    Qian, A R; Li, D; Han, J; Gao, X; Di, S M; Zhang, W; Hu, L F; Shang, Peng

    2012-05-01

    Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

  19. 3D-printed and CNC milled flow-cells for chemiluminescence detection.

    Science.gov (United States)

    Spilstead, Kara B; Learey, Jessica J; Doeven, Egan H; Barbante, Gregory J; Mohr, Stephan; Barnett, Neil W; Terry, Jessica M; Hall, Robynne M; Francis, Paul S

    2014-08-01

    Herein we explore modern fabrication techniques for the development of chemiluminescence detection flow-cells with features not attainable using the traditional coiled tubing approach. This includes the first 3D-printed chemiluminescence flow-cells, and a milled flow-cell designed to split the analyte stream into two separate detection zones within the same polymer chip. The flow-cells are compared to conventional detection systems using flow injection analysis (FIA) and high performance liquid chromatography (HPLC), with the fast chemiluminescence reactions of an acidic potassium permanganate reagent with morphine and a series of adrenergic phenolic amines.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 3D Printing of Scaffold for Cells Delivery: Advances in Skin Tissue Engineering

    Directory of Open Access Journals (Sweden)

    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.

  2. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    Science.gov (United States)

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

  3. 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood.

    Science.gov (United States)

    Zheng, Siyang; Lin, Henry K; Lu, Bo; Williams, Anthony; Datar, Ram; Cote, Richard J; Tai, Yu-Chong

    2011-02-01

    Detection of circulating tumor cells has emerged as a promising minimally invasive diagnostic and prognostic tool for patients with metastatic cancers. We report a novel three dimensional microfilter device that can enrich viable circulating tumor cells from blood. This device consists of two layers of parylene membrane with pores and gap precisely defined with photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimize the stress concentration on cell membrane and sustain cell viability during filtration. Viable cell capture on device was investigated with scanning electron microscopy, confocal microscopy, and immunofluorescent staining using model systems of cultured tumor cells spiked in blood or saline. The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application. The device provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings.

  4. Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix.

    Science.gov (United States)

    Pedersen, John A; Boschetti, Federica; Swartz, Melody A

    2007-01-01

    Interstitial fluid flow has been shown to affect the organization and behavior of cells in 3D environments in vivo and in vitro, yet the forces driving such responses are not clear. Due to the complex architecture of the extracellular matrix (ECM) and the difficulty of measuring fluid flow near cells embedded in it, the levels of shear stress experienced by cells in this environment are typically estimated using bulk-averaged matrix parameters such as hydraulic permeability. While this is useful for estimating average stresses, it cannot yield insight into how local matrix fiber architecture-which is cell-controlled in the immediate pericellular environment-affects the local stresses imposed on the cell surface. To address this, we used computational fluid dynamics to study flow through an idealized mesh constructed of a cubic lattice of fibers simulating a typical in vitro collagen gel. We found that, in such high porosity matrices, the fibers strongly affect the flow fields near the cell, with peak shear stresses up to five times higher than those predicted by the Brinkman equation. We also found that minor remodeling of the fibers near the cell surface had major effects on the shear stress profile on the cell. These findings demonstrate the importance of fiber architecture to the fluid forces on a cell embedded in a 3D matrix, and also show how small modifications in the local ECM can lead to large changes in the mechanical environment of the cell.

  5. The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology.

    Science.gov (United States)

    Zhao, Yu; Li, Yang; Mao, Shuangshuang; Sun, Wei; Yao, Rui

    2015-11-02

    Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability and cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for 3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks.

  6. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

    Science.gov (United States)

    Radtke, Andrea L; Herbst-Kralovetz, Melissa M

    2012-04-03

    Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties

  7. Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

    Science.gov (United States)

    Kurniawan, Nicholas Agung; Chaudhuri, Parthiv Kant; Lim, Chwee Teck

    2015-01-01

    The ability of cells to migrate is crucial in a wide variety of cell functions throughout life from embryonic development and wound healing to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell’s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration. PMID:25867104

  8. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Science.gov (United States)

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

    2016-01-01

    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 cm(2) 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.

  9. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    Science.gov (United States)

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-03-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work.

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

    CERN Document Server

    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.

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

    CERN Document Server

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. High Throughput Studies of Cell Migration in 3D Microtissues Fabricated by a Droplet Microfluidic Chip

    Directory of Open Access Journals (Sweden)

    Xiangchen Che

    2016-05-01

    Full Text Available Arrayed three-dimensional (3D micro-sized tissues with encapsulated cells (microtissues have been fabricated by a droplet microfluidic chip. The extracellular matrix (ECM is a polymerized collagen network. One or multiple breast cancer cells were embedded within the microtissues, which were stored in arrayed microchambers on the same chip without ECM droplet shrinkage over 48 h. The migration trajectory of the cells was recorded by optical microscopy. The migration speed was calculated in the range of 3–6 µm/h. Interestingly, cells in devices filled with a continuous collagen network migrated faster than those where only droplets were arrayed in the chambers. This is likely due to differences in the length scales of the ECM network, as cells embedded in thin collagen slabs also migrate slower than those in thick collagen slabs. In addition to migration, this technical platform can be potentially used to study cancer cell-stromal cell interactions and ECM remodeling in 3D tumor-mimicking environments.

  14. Limbal melanocytes support limbal epithelial stem cells in 2D and 3D microenvironments.

    Science.gov (United States)

    Dziasko, Marc A; Tuft, Stephen J; Daniels, Julie T

    2015-09-01

    Human limbal epithelial stem cells (LESCs) are essential for the maintenance of the corneal epithelium of the ocular surface. LESCs are located within limbal crypts between the palisades of Vogt in the limbus; the interface between the peripheral cornea and conjunctiva. The limbal crypts have been proposed as a LESC niche owing to their support of epithelial cells, which can form holoclone colonies in vitro. Closely associated with the limbal crypts is a concentrated population of melanocytes. The anatomical location and close proximity to putative LESC suggests that melanocytes might play a role in maintenance of these stem cells in the niche. The aim of this study was to assess the ability of human limbal melanocytes (hLM) to support the expansion of human limbal epithelial cells (LECs) in vitro as an indicator of functional cell-cell interaction. After observing that hLM co-localize with clusters of compact epithelial cells in the native limbal crypts, hLM were isolated from crypt-rich cadaveric limbal biopsies and used as feeders for the culture of LECs. Interestingly, LECs grown on mitotically active hLM were able to generate large epithelial colonies that contained small and compact cells with morphological stem cell characteristics. Immunocytochemistry revealed that LECs expanded on hLM were positive for the expression of the putative stem cell markers CK15, Bmi-1 and p63α and negative for the marker of terminal cell differentiation CK3. LECs and hLM were finally co-cultured on RAFT (real architecture for 3D tissue) collagen tissue equivalents. In 3D co-cultures, hLM promoted multi-layering of the epithelial sheet in which basal cells were maintained in an undifferentiated state. Taken together, these observations suggest melanocytes could play an important role in the maintenance of LESCs in the native human limbal stem cell niche.

  15. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Investigation of T-cell receptor-γ gene rearrangement in gastrointestinal lymphomas by PCR-SSCP analysis

    Institute of Scientific and Technical Information of China (English)

    Xi-Qun Han; Li He; Lan-Ying Shong; Hui-Yong Jiang; Mei-Gang Zhu; Tong Zhao

    2004-01-01

    AIM: To analyze the characterization of T-cell receptor-γ (TCR-γ) gene rearrangement in the gastrointestinal lymphomas and evaluate the value of PCR-SSCP analysis in gastrointestinal lymphomas investigation.METHODS: TCR-γgene rearrangement segments of gastrointestinal lymphomas were cloned and sequenced.Single clone plasmid and mixed clone plsamids were subsequently submitted to PCR-SSCP analysis to investigate the relationship between the number of amplified clones and band patterns of the amplified products. The PCR products of TCR-γgene rearrangement of 40 gastrointestinal lymphomas were electrophoresed on agarose gels and the positive cases on agarose gels were studied by SSCP analysis.RESULTS: The sequencing showed that TCR-γ gene rearrangement of the gastrointestinal lymphomas included functional gene and pseudogene with extensive variety in the junctional regions. In SSCP analysis, the number of the single-stranded bands was about two times of the number of amplified clones, and double-stranded band became broad with the increased number of the amplified clones. Thirteen of the 25 B-cell gastrointestinal lymphomas and 14 of the 15 gastrointestinal T-cell lymphomas were positive detected on agarose gel electrophoresis. Of the positive cases detected by SSCP analysis, 3 B-cell lymphomas and 13 T-cell lymphomas showed positive bands. The other cases showed only smears. The rearranged pattern included 13 monoallelic gene rearrangements and 3 biallelic or oligoclonal gene rearrangements.CONCLUSION: The pattern of TCR-γ, gene rearrangement in gastrointestinal lymphomas are similar to that of the nodular lymphomas. PCR-SSCP analysis for TCR-γ gene rearrangement can be applied both for adjuvant diagnosis of gastrointestinal lymphomas and analysis of the gene rearrangement pattern. The ratio of TCR-γ gene rearrangements occurred in T-cell gastrointestinal lymphomas is significantly higher than that in B-cell gastrointestinal lymphomas. The gene rearrangement

  18. Image informatics for studying signal transduction in cells interacting with 3D matrices

    Science.gov (United States)

    Tzeranis, Dimitrios S.; Guo, Jin; Chen, Chengpin; Yannas, Ioannis V.; Wei, Xunbin; So, Peter T. C.

    2014-03-01

    Cells sense and respond to chemical stimuli on their environment via signal transduction pathways, complex networks of proteins whose interactions transmit chemical information. This work describes an implementation of image informatics, imaging-based methodologies for studying signal transduction networks. The methodology developed focuses on studying signal transduction networks in cells that interact with 3D matrices. It utilizes shRNA-based knock down of network components, 3D high-content imaging of cells inside the matrix by spectral multi-photon microscopy, and single-cell quantification using features that describe both cell morphology and cell-matrix adhesion pattern. The methodology is applied in a pilot study of TGFβ signaling via the SMAD pathway in fibroblasts cultured inside porous collagen-GAG scaffolds, biomaterials similar to the ones used clinically to induce skin regeneration. Preliminary results suggest that knocking down all rSMAD components affects fibroblast response to TGFβ1 and TGFβ3 isoforms in different ways, and suggest a potential role for SMAD1 and SMAD5 in regulating TGFβ isoform response. These preliminary results need to be verified with proteomic results that can provide solid evidence about the particular role of individual components of the SMAD pathway.

  19. Impact of dimensionality and network disruption on microrheology of cancer cells in 3D environments.

    Directory of Open Access Journals (Sweden)

    Michael Mak

    2014-11-01

    Full Text Available Dimensionality is a fundamental component that can have profound implications on the characteristics of physical systems. In cell biology, however, the majority of studies on cell physical properties, from rheology to force generation to migration, have been performed on 2D substrates, and it is not clear how a more realistic 3D environment influences cell properties. Here, we develop an integrated approach and demonstrate the combination of mitochondria-tracking microrheology, microfluidics, and Brownian dynamics simulations to explore the impact of dimensionality on intracellular mechanics and on the effects of intracellular disruption. Additionally, we consider both passive thermal and active motor-driven processes within the cell and demonstrate through modeling how active internal fluctuations are modulated via dimensionality. Our results demonstrate that metastatic breast cancer cells (MDA-MB-231 exhibit more solid-like internal motions in 3D compared to 2D, and actin network disruption via Cytochalasin D has a more pronounced effect on internal cell fluctuations in 2D. Our computational results and modeling show that motor-induced active stress fluctuations are enhanced in 2D, leading to increased local intracellular particle fluctuations and apparent fluid-like behavior.

  20. Reconstruction of 3D ion beam micro-tomography data for applications in Cell Biology

    Energy Technology Data Exchange (ETDEWEB)

    Habchi, C. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France)], E-mail: habchi@cenbg.in2p3.fr; Nguyen, D.T.; Barberet, Ph. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Incerti, S. [CNRS/IN2P3, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Moretto, Ph. [Universite de Bordeaux, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France); Sakellariou, A. [Research School of Physical Sciences and Engineering, Australian National University, Canberra ACT 0200 (Australia); Seznec, H. [CNRS/IN2P3, Centre d' Etudes Nucleaires de Bordeaux Gradignan, UMR 5797, Gradignan F-33175 (France)

    2009-06-15

    The DISRA (Discrete Image Space Reconstruction Algorithm) reconstruction code, created by A. Sakellariou, was conceived for the ideal case of complete three-dimensional (3D) PIXET (Particle Induced X-ray Emission Tomography) data. This implies two major difficulties for biological samples: first, the long duration of such experiments and second, the subsequent damage that occurs on such fragile specimens. For this reason, the DISRA code was extended at CENBG in order to probe isolated PIXET slices, taking into account the sample structure and mass density provided by 3D STIMT (Scanning Transmission Ion Microscopy Tomography) in the volume of interest. This modified version was tested on a phantom sample and first results on human cancer cells are also presented.

  1. Reconstruction of 3D ion beam micro-tomography data for applications in Cell Biology

    Science.gov (United States)

    Habchi, C.; Nguyen, D. T.; Barberet, Ph.; Incerti, S.; Moretto, Ph.; Sakellariou, A.; Seznec, H.

    2009-06-01

    The DISRA (Discrete Image Space Reconstruction Algorithm) reconstruction code, created by A. Sakellariou, was conceived for the ideal case of complete three-dimensional (3D) PIXET (Particle Induced X-ray Emission Tomography) data. This implies two major difficulties for biological samples: first, the long duration of such experiments and second, the subsequent damage that occurs on such fragile specimens. For this reason, the DISRA code was extended at CENBG in order to probe isolated PIXET slices, taking into account the sample structure and mass density provided by 3D STIMT (Scanning Transmission Ion Microscopy Tomography) in the volume of interest. This modified version was tested on a phantom sample and first results on human cancer cells are also presented.

  2. Flow-through 3D biofuel cell anode for NAD{sup +}-dependent enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Rincon, Rosalba A.; Lau, Carolin; Garcia, Kristen E. [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States); Atanassov, Plamen, E-mail: plamen@unm.ed [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States)

    2011-02-01

    NAD{sup +}-dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD{sup +}-dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan; allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Neufeld Gera

    2009-11-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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

  7. Tracking immune-related cell responses to drug delivery microparticles in 3D dense collagen matrix.

    Science.gov (United States)

    Obarzanek-Fojt, Magdalena; Curdy, Catherine; Loggia, Nicoletta; Di Lena, Fabio; Grieder, Kathrin; Bitar, Malak; Wick, Peter

    2016-10-01

    Beyond the therapeutic purpose, the impact of drug delivery microparticles on the local tissue and inflammatory responses remains to be further elucidated specifically for reactions mediated by the host immune cells. Such immediate and prolonged reactions may adversely influence the release efficacy and intended therapeutic pathway. The lack of suitable in vitro platforms limits our ability to gain insight into the nature of immune responses at a single cell level. In order to establish an in vitro 3D system mimicking the connective host tissue counterpart, we utilized reproducible, compressed, rat-tail collagen polymerized matrices. THP1 cells (human acute monocytic leukaemia cells) differentiated into macrophage-like cells were chosen as cell model and their functionality was retained in the dense rat-tail collagen matrix. Placebo microparticles were later combined in the immune cell seeded system during collagen polymerization and secreted pro-inflammatory factors: TNFα and IL-8 were used as immune response readout (ELISA). Our data showed an elevated TNFα and IL-8 secretion by macrophage THP1 cells indicating that Placebo microparticles trigger certain immune cell responses under 3D in vivo like conditions. Furthermore, we have shown that the system is sensitive to measure the differences in THP1 macrophage pro-inflammatory responses to Active Pharmaceutical Ingredient (API) microparticles with different API release kinetics. We have successfully developed a tissue-like, advanced, in vitro system enabling selective "readouts" of specific responses of immune-related cells. Such system may provide the basis of an advanced toolbox enabling systemic evaluation and prediction of in vivo microparticle reactions on human immune-related cells.

  8. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it po

  9. Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells.

    Directory of Open Access Journals (Sweden)

    Mai Nanya

    Full Text Available Etoposide, a topoisomerase 2 (TOP2 inhibitor, is associated with the development of KMT2A (MLL-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.

  10. Fabrication of 3-D Reconstituted Organoid Arrays by DNA-Programmed Assembly of Cells (DPAC).

    Science.gov (United States)

    Todhunter, Michael E; Weber, Robert J; Farlow, Justin; Jee, Noel Y; Cerchiari, Alec E; Gartner, Zev J

    2016-09-13

    Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three-dimensional (3-D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA-programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3-D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide "Velcro," allowing rapid, specific, and reversible cell adhesion to a two-dimensional (2-D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer-by-layer, from this initial 2-D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids and permits positioning of constituent cells with single-cell resolution even within cultures several centimeters long. © 2016 by John Wiley & Sons, Inc.

  11. Current automated 3D cell detection methods are not a suitable replacement for manual stereologic cell counting

    Directory of Open Access Journals (Sweden)

    Christoph eSchmitz

    2014-05-01

    Full Text Available Stereologic cell counting has had a major impact on the field of neuroscience. A major bottleneck in stereologic cell counting is that the user must manually decide whether or not each cell is counted according to three-dimensional (3D stereologic counting rules by visual inspection within hundreds of microscopic fields-of-view per investigated brain or brain region. Reliance on visual inspection forces stereologic cell counting to be very labor-intensive and time-consuming, and is the main reason why biased, non-stereologic two-dimensional (2D cell counting approaches have remained in widespread use. We present an evaluation of the performance of modern automated cell detection and segmentation algorithms as a potential alternative to the manual approach in stereologic cell counting. The image data used in this study were 3D microscopic images of thick brain tissue sections prepared with a variety of commonly used nuclear and cytoplasmic stains. The evaluation compared the numbers and locations of cells identified unambiguously and counted exhaustively by an expert observer with those found by three automated 3D cell detection algorithms: nuclei segmentation from the FARSIGHT toolkit, nuclei segmentation by 3D multiple level set methods, and the 3D object counter plug-in for ImageJ. Of these methods, FARSIGHT performed best, with true-positive detection rates between 38–99% and false-positive rates from 3.6–82%. The results demonstrate that the current automated methods suffer from lower detection rates and higher false-positive rates than are acceptable for obtaining valid estimates of cell numbers. Thus, at present, stereologic cell counting with manual decision for object inclusion according to unbiased stereologic counting rules remains the only adequate method for unbiased cell quantification in histologic tissue sections.

  12. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    Science.gov (United States)

    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.

  13. Fabrication of solution processed 3D nanostructured CuInGaS₂ thin film solar cells.

    Science.gov (United States)

    Chu, Van Ben; Cho, Jin Woo; Park, Se Jin; Hwang, Yun Jeong; Park, Hoo Keun; Do, Young Rag; Min, Byoung Koun

    2014-03-28

    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.

  14. 3D cut-cell modelling for high-resolution atmospheric simulations

    CERN Document Server

    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...

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

    Science.gov (United States)

    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.

  16. 3D bioprinting of biomimetic aortic vascular constructs with self-supporting cells.

    Science.gov (United States)

    Kucukgul, Can; Ozler, S Burce; Inci, Ilyas; Karakas, Ezgi; Irmak, Ster; Gozuacik, Devrim; Taralp, Alpay; Koc, Bahattin

    2015-04-01

    Cardiovascular diseases are the leading cause of deaths throughout the world. Vascular diseases are mostly treated with autografts and blood vessel transplantations. However, traditional grafting methods have several problems including lack of suitable harvest sites, additional surgical costs for harvesting procedure, pain, infection, lack of donors, and even no substitutes at all. Recently, tissue engineering and regenerative medicine approaches are used to regenerate damaged or diseased tissues. Most of the tissue engineering investigations have been based on the cell seeding into scaffolds by providing a suitable environment for cell attachment, proliferation, and differentiation. Because of the challenges such as difficulties in seeding cells spatially, rejection, and inflammation of biomaterials used, the recent tissue engineering studies focus on scaffold-free techniques. In this paper, the development of novel computer aided algorithms and methods are developed for 3D bioprinting of scaffold-free biomimetic macrovascular structures. Computer model mimicking a real human aorta is generated using imaging techniques and the proposed computational algorithms. An optimized three-dimensional bioprinting path planning are developed with the proposed self-supported model. Mouse embryonic fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D bioprinted layer-by-layer according to the proposed self-supported method to form an aortic tissue construct.

  17. An automated tool for 3D tracking of single molecules in living cells

    Science.gov (United States)

    Gardini, L.; Capitanio, M.; Pavone, F. S.

    2015-07-01

    Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. Since the behaviour of proteins and biological molecules is tightly influenced by the cell's environment, a growing number of microscopy techniques are moving from in vitro to live cell experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). To satisfy these requirements we developed an automated routine that allow 3D tracking of single fluorescent molecules in living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.

  18. Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

    Science.gov (United States)

    Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

    2016-01-01

    Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment.

  19. Construction of 3D micropatterned surfaces with wormlike and superhydrophilic PEG brushes to detect dysfunctional cells.

    Science.gov (United States)

    Hou, Jianwen; Shi, Qiang; Ye, Wei; Fan, Qunfu; Shi, Hengchong; Wong, Shing-Chung; Xu, Xiaodong; Yin, Jinghua

    2014-12-10

    Detection of dysfunctional and apoptotic cells plays an important role in clinical diagnosis and therapy. To develop a portable and user-friendly platform for dysfunctional and aging cell detection, we present a facile method to construct 3D patterns on the surface of styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS) with poly(ethylene glycol) brushes. Normal red blood cells (RBCs) and lysed RBCs (dysfunctional cells) are used as model cells. The strategy is based on the fact that poly(ethylene glycol) brushes tend to interact with phosphatidylserine, which is in the inner leaflet of normal cell membranes but becomes exposed in abnormal or apoptotic cell membranes. We demonstrate that varied patterned surfaces can be obtained by selectively patterning atom transfer radical polymerization (ATRP) initiators on the SEBS surface via an aqueous-based method and growing PEG brushes through surface-initiated atom transfer radical polymerization. The relatively high initiator density and polymerization temperature facilitate formation of PEG brushes in high density, which gives brushes worm-like morphology and superhydrophilic property; the tendency of dysfunctional cells adhered on the patterned surfaces is completely different from well-defined arrays of normal cells on the patterned surfaces, providing a facile method to detect dysfunctional cells effectively. The PEG-patterned surfaces are also applicable to detect apoptotic HeLa cells. The simplicity and easy handling of the described technique shows the potential application in microdiagnostic devices.

  20. Rapid, Nonradioactive Detection of Clonal T-Cell Receptor Gene Rearrangements in Lymphoid Neoplasms

    Science.gov (United States)

    Bourguin, Anne; Tung, Rosann; Galili, Naomi; Sklar, Jeffrey

    1990-11-01

    Southern blot hybridization analysis of clonal antigen receptor gene rearrangements has proved to be a valuable adjunct to conventional methods for diagnosing lymphoid neoplasia. However, Southern blot analysis suffers from a number of technical disadvantages, including the time necessary to obtain results, the use of radioactivity, and the susceptibility of the method to various artifacts. We have investigated an alternative approach for assessing the clonality of antigen receptor gene rearrangements in lymphoid tissue biopsy specimens. This approach involves the amplification of rearranged γ T-cell receptor genes by the polymerase chain reaction and analysis of the polymerase chain reaction products by denaturing gradient gel electrophoresis. By use of this approach, clonal rearrangements from neoplastic lymphocytes constituting as little as 0.1-1% of the total cells in the tissue are detected as discrete bands in the denaturing gel after the gel is stained with ethidium bromide and viewed under ultraviolet light. In contrast, polyclonal rearrangements from reactive lymphocytes appear as a diffuse smear along the length of the gel. Our findings suggest that polymerase chain reaction combined with denaturing gradient gel electrophoresis may offer a rapid, nonradioactive, and sensitive alternative to Southern blot analysis for the diagnostic evaluation of lymphoid tissue biopsy specimens.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    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......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...

  3. Brownian nanoimaging of interface dynamics and ligand-receptor binding at cell surfaces in 3-D.

    Science.gov (United States)

    Kuznetsov, Igor R; Evans, Evan A

    2013-04-01

    We describe a method for nanoimaging interfacial dynamics and ligand-receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-μm diameter glass bead confined by a soft laser trap to create a "cloud" of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ~10 ms intervals with bare precisions (±SD) of 4-6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surface receptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ~1 nm.

  4. Tuning 3D Collagen Matrix Stiffness Independently of Collagen Concentration Modulates Endothelial Cell Behavior

    Science.gov (United States)

    Mason, Brooke N.; Starchenko, Alina; Williams, Rebecca M.; Bonassar, Lawrence J.; Reinhart-King, Cynthia A.

    2012-01-01

    Numerous studies have described the effects of matrix stiffening on cell behavior using two dimensional (2D) synthetic surfaces; however less is known about the effects of matrix stiffening on cells embedded in three dimensional (3D) in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in 3D is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a 3-fold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation endproducts is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth. PMID:22902816

  5. Scaffolds fabricated by 3D two-photon photopolymerization for live cell studies

    Science.gov (United States)

    Teplicky, T.; Cunderlikova, B.; Mateasik, A.; Vincze, A.; Chorvat, D.; Marcek Chorvatova, A.

    2016-12-01

    Design and fabrication of appropriate biocompatible microstructures that ensure fixation and control of experimental conditions for live cell and bacteria observations is an important prerequisite for number of real time experiments. Our approach is to design engineered microfabricated 3D structures for growth of cells in culture without significant modification of their metabolic state. Presented approach is aimed at evaluation of the potential applicability of biocompatible constructs in the biomedical field and thus live cell monitoring in controlled conditions. Design and evaluation of properties of materials and structures with mesoscopic arrangement and their interaction with biological objects is a prerequisite for establishment of physiologically relevant in vitro models of pathologies as well as for development of a new generation of nano / micro / bio-sensors.

  6. [Analyses of the rearrangement of T-cell receptor- and immunoglobulin genes in the diagnosis of lymphoproliferative disorders].

    Science.gov (United States)

    Griesser, D H

    1995-01-01

    Rearrangements are developmentally regulated genetic recombinations in T and B cells which generate functional T cell receptor (TcR) and immunoglobulin genes, respectively. Different variable, sometimes diversity, and joining gene segments which are discontinuously spread out within their chromosomal location in germline configuration, are randomly assembled in individual lymphocytes. These rearrangements can be detected by Southern Blot analysis if more than 5% of a total lymphocyte population in a biopsy specimen carries the same clonal rearrangement. We analyzed DNA from 324 snap-frozen biopsy specimens from lympho-proliferative disorders. None of the 20 reactive lesions and four malignant myelomonocytic tumors had a clonal antigen receptor gene rearrangement. All 117 malignant B cell lymphomas of different subtypes and 95 of 97 malignant T cell lymphomas showed a clonal gene rearrangement. Only two angioimmunoblastic lymphadenopathy(AILD)-type T cell lymphomas did not have immune receptor gene rearrangements. They were morphologically indistinguishable from the other 47 T/AILD lymphomas with clonal rearrangement patterns. In most cases TcR beta and immunoglobulin heavy chain (IgH) gene probes were sufficient for lineage assignment of the clonal T or B lymphocyte population. In 18% of B lymphomas, however, a cross-lineage rearrangement of TcR beta genes, and in 20% of the T cell lymphomas a clonal IgH gene rearrangement was detected. After exclusion of centrocytic, large cell anaplastic lymphomas (LCAL) of B-type, and T/AILD lymphomas which are overrepresented in our study, only 10% of the remaining 147 T and B cell lymphomas had aberrant rearrangements. TcR rearrangements other than those of the beta chain genes were extremely rare in B cell lymphomas, as were Ig kappa rearrangements in T lymphomas. Only two T/AILD lymphomas had IgH and Ig kappa rearrangement in addition to their clonal T cell receptor gene rearrangements. Both samples likely contain a clonal B

  7. AC electric field induced dipole-based on-chip 3D cell rotation.

    Science.gov (United States)

    Benhal, Prateek; Chase, J Geoffrey; Gaynor, Paul; Oback, Björn; Wang, Wenhui

    2014-08-01

    The precise rotation of suspended cells is one of the many fundamental manipulations used in a wide range of biotechnological applications such as cell injection and enucleation in nuclear transfer (NT) cloning. Noticeably scarce among the existing rotation techniques is the three-dimensional (3D) rotation of cells on a single chip. Here we present an alternating current (ac) induced electric field-based biochip platform, which has an open-top sub-mm square chamber enclosed by four sidewall electrodes and two bottom electrodes, to achieve rotation about the two axes, thus 3D cell rotation. By applying an ac potential to the four sidewall electrodes, an in-plane (yaw) rotating electric field is generated and in-plane rotation is achieved. Similarly, by applying an ac potential to two opposite sidewall electrodes and the two bottom electrodes, an out-of-plane (pitch) rotating electric field is generated and rolling rotation is achieved. As a prompt proof-of-concept, bottom electrodes were constructed with transparent indium tin oxide (ITO) using the standard lift-off process and the sidewall electrodes were constructed using a low-cost micro-milling process and then assembled to form the chip. Through experiments, we demonstrate rotation of bovine oocytes of ~120 μm diameter about two axes, with the capability of controlling the rotation direction and the rate for each axis through control of the ac potential amplitude, frequency, and phase shift, and cell medium conductivity. The maximum observed rotation rate reached nearly 140° s⁻¹, while a consistent rotation rate reached up to 40° s⁻¹. Rotation rate spectra for zona pellucida-intact and zona pellucida-free oocytes were further compared and found to have no effective difference. This simple, transparent, cheap-to-manufacture, and open-top platform allows additional functional modules to be integrated to become a more powerful cell manipulation system.

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

    Directory of Open Access Journals (Sweden)

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

    Science.gov (United States)

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

    2016-06-07

    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

  10. Laser irradiated fluorescent perfluorocarbon microparticles in 2-D and 3-D breast cancer cell models

    Science.gov (United States)

    Niu, Chengcheng; Wang, Long; Wang, Zhigang; Xu, Yan; Hu, Yihe; Peng, Qinghai

    2017-01-01

    Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work. PMID:28262671

  11. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    Science.gov (United States)

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  12. Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

    Science.gov (United States)

    Esch, Mandy B; Prot, Jean-Matthieu; Wang, Ying I; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A; Applegate, Dawn R; Shuler, Michael L

    2015-05-21

    We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation

  13. High power density microbial fuel cell with flexible 3D graphene-nickel foam as anode

    Science.gov (United States)

    Wang, Hanyu; Wang, Gongming; Ling, Yichuan; Qian, Fang; Song, Yang; Lu, Xihong; Chen, Shaowei; Tong, Yexiang; Li, Yat

    2013-10-01

    The structure and electrical conductivity of anode play a significant role in the power generation of microbial fuel cells (MFCs). In this study, we developed a three-dimensional (3D) reduced graphene oxide-nickel (denoted as rGO-Ni) foam as an anode for MFC through controlled deposition of rGO sheets onto the nickel foam substrate. The loading amount of rGO sheets and electrode surface area can be controlled by the number of rGO loading cycles. 3D rGO-Ni foam anode provides not only a large accessible surface area for microbial colonization and electron mediators, but also a uniform macro-porous scaffold for effective mass diffusion of the culture medium. Significantly, at a steady state of the power generation, the MFC device with flexible rGO-Ni electrodes produced an optimal volumetric power density of 661 W m-3 calculated based on the volume of anode material, or 27 W m-3 based on the volume of the anode chamber. These values are substantially higher than that of plain nickel foam, and other conventional carbon based electrodes (e.g., carbon cloth, carbon felt, and carbon paper) measured in the same conditions. To our knowledge, this is the highest volumetric power density reported for mL-scale MFC device with a pure strain of Shewanella oneidensis MR-1. We also demonstrated that the MFC device can be operated effectively in a batch-mode at least for a week. These new 3D rGO-Ni electrodes show great promise for improving the power generation of MFC devices.The structure and electrical conductivity of anode play a significant role in the power generation of microbial fuel cells (MFCs). In this study, we developed a three-dimensional (3D) reduced graphene oxide-nickel (denoted as rGO-Ni) foam as an anode for MFC through controlled deposition of rGO sheets onto the nickel foam substrate. The loading amount of rGO sheets and electrode surface area can be controlled by the number of rGO loading cycles. 3D rGO-Ni foam anode provides not only a large accessible

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

    Science.gov (United States)

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

    2016-02-04

    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.

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

    Science.gov (United States)

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

    2016-05-24

    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).

  16. Characterization of Porcine Ventral Mesencephalic Precursor Cells following Long-Term Propagation in 3D Culture

    Directory of Open Access Journals (Sweden)

    Pia S. Jensen

    2012-01-01

    Full Text Available The potential use of predifferentiated neural precursor cells for treatment of a neurological disorder like Parkinson’s disease combines stem cell research with previous experimental and clinical transplantation of developing dopaminergic neurons. One current obstacle is, however, the lack of ability to generate dopaminergic neurons after long-term in vitro propagation of the cells. The domestic pig is considered a useful nonprimate large animal model in neuroscience, because of a better resemblance of the larger gyrencephalic pig brain to the human brain than the commonly used brains of smaller rodents. In the present study, porcine embryonic (28–30 days, ventral mesencephalic precursor cells were isolated and propagated as free-floating neural tissue spheres in medium containing epidermal growth factor and fibroblast growth factor 2. For passaging, the tissue spheres were cut into quarters, avoiding mechanical or enzymatic dissociation in order to minimize cellular trauma and preserve intercellular contacts. Spheres were propagated for up to 237 days with analysis of cellular content and differentiation at various time points. Our study provides the first demonstration that porcine ventral mesencephalic precursor cells can be long-term propagated as neural tissue spheres, thereby providing an experimental 3D in vitro model for studies of neural precursor cells, their niche, and differentiation capacity.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  19. Pico-projector-based optical sectioning microscopy for 3D chlorophyll fluorescence imaging of mesophyll cells

    Science.gov (United States)

    Chen, Szu-Yu; Hsu, Yu John; Yeh, Chia-Hua; Chen, S.-Wei; Chung, Chien-Han

    2015-03-01

    A pico-projector-based optical sectioning microscope (POSM) was constructed using a pico-projector to generate structured illumination patterns. A net rate of 5.8 × 106 pixel/s and sub-micron spatial resolution in three-dimensions (3D) were achieved. Based on the pico-projector’s flexibility in pattern generation, the characteristics of POSM with different modulation periods and at different imaging depths were measured and discussed. With the application of different modulation periods, 3D chlorophyll fluorescence imaging of mesophyll cells was carried out in freshly plucked leaves of four species without sectioning or staining. For each leaf, an average penetration depth of 120 μm was achieved. Increasing the modulation period along with the increment of imaging depth, optical sectioning images can be obtained with a compromise between the axial resolution and signal-to-noise ratio. After ∼30 min imaging on the same area, photodamage was hardly observed. Taking the advantages of high speed and low damages of POSM, the investigation of the dynamic fluorescence responses to temperature changes was performed under three different treatment temperatures. The three embedded blue, green and red light-emitting diode light sources were applied to observe the responses of the leaves with different wavelength excitation.

  20. 2D/3D perovskite hybrids as moisture-tolerant and efficient light absorbers for solar cells.

    Science.gov (United States)

    Ma, Chaoyan; Leng, Chongqian; Ji, Yixiong; Wei, Xingzhan; Sun, Kuan; Tang, Linlong; Yang, Jun; Luo, Wei; Li, Chaolong; Deng, Yunsheng; Feng, Shuanglong; Shen, Jun; Lu, Shirong; Du, Chunlei; Shi, Haofei

    2016-11-03

    The lifetime and power conversion efficiency are the key issues for the commercialization of perovskite solar cells (PSCs). In this paper, the development of 2D/3D perovskite hybrids (CA2PbI4/MAPbIxCl3-x) was firstly demonstrated to be a reliable method to combine their advantages, and provided a new concept for achieving both stable and efficient PSCs through the hybridization of perovskites. 2D/3D perovskite hybrids afforded significantly-improved moisture stability of films and devices without encapsulation in a high humidity of 63 ± 5%, as compared with the 3D perovskite (MAPbIxCl3-x). The 2D/3D perovskite-hybrid film did not undergo any degradation after 40 days, while the 3D perovskite decomposed completely under the same conditions after 8 days. The 2D/3D perovskite-hybrid device maintained 54% of the original efficiency after 220 hours, whereas the 3D perovskite device lost all the efficiency within only 50 hours. Moreover, the 2D/3D perovskite hybrid achieved comparable device performances (PCE: 13.86%) to the 3D perovskite (PCE: 13.12%) after the optimization of device fabrication conditions.

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

    Directory of Open Access Journals (Sweden)

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

    Science.gov (United States)

    Chang, Chih-Hao; Lin, Chih-Yang; Liu, Fwu-Hsing; Chen, Mark Hung-Chih; Lin, Chun-Pin; Ho, Hong-Nerng; Liao, Yunn-Shiuan

    2015-01-01

    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.

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

    Science.gov (United States)

    Chang, Chih-Hao; Lin, Chih-Yang; Liu, Fwu-Hsing; Chen, Mark Hung-Chih; Lin, Chun-Pin; Ho, Hong-Nerng; Liao, Yunn-Shiuan

    2015-01-01

    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. PMID:26618362

  4. Characterization of gene rearrangements resulted from genomic structural aberrations in human esophageal squamous cell carcinoma KYSE150 cells.

    Science.gov (United States)

    Hao, Jia-Jie; Gong, Ting; Zhang, Yu; Shi, Zhi-Zhou; Xu, Xin; Dong, Jin-Tang; Zhan, Qi-Min; Fu, Song-Bin; Wang, Ming-Rong

    2013-01-15

    Chromosomal rearrangements and involved genes have been reported to play important roles in the development and progression of human malignancies. But the gene rearrangements in esophageal squamous cell carcinoma (ESCC) remain to be identified. In the present study, array-based comparative genomic hybridization (array-CGH) was performed on the ESCC cell line KYSE150. Eight disrupted genes were detected according to the obviously distinct unbalanced breakpoints. The splitting of these genes was validated by dual-color fluorescence in-situ hybridization (FISH). By using rapid amplification of cDNA ends (RACE), genome walking and sequencing analysis, we further identified gene disruptions and rearrangements. A fusion transcript DTL-1q42.2 was derived from an intrachromosomal rearrangement of chromosome 1. Highly amplified segments of DTL and PTPRD were self-rearranged. The sequences on either side of the junctions possess micro-homology with each other. FISH results indicated that the split DTL and PTPRD were also involved in comprising parts of the derivative chromosomes resulted from t(1q;9p;12p) and t(9;1;9). Further, we found that regions harboring DTL (1q32.3) and PTPRD (9p23) were also splitting in ESCC tumors. The data supplement significant information on the existing genetic background of KYSE150, which may be used as a model for studying these gene rearrangements.

  5. Label-free optical detection of cells grown in 3D silicon microstructures.

    Science.gov (United States)

    Merlo, Sabina; Carpignano, Francesca; Silva, Gloria; Aredia, Francesca; Scovassi, A Ivana; Mazzini, Giuliano; Surdo, Salvatore; Barillaro, Giuseppe

    2013-08-21

    We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

  6. Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging.

    Science.gov (United States)

    Annibale, Paolo; Gratton, Enrico

    2015-03-19

    Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell, demonstrating a correlation between local transcription kinetics and the movement of the transcription site. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability.

  7. Identifying same-cell contours in image stacks: a key step in making 3D reconstructions.

    Science.gov (United States)

    Leung, Tony Kin Shun; Veldhuis, Jim H; Krens, S F Gabby; Heisenberg, C P; Brodland, G Wayne

    2011-02-01

    Identification of contours belonging to the same cell is a crucial step in the analysis of confocal stacks and other image sets in which cell outlines are visible, and it is central to the making of 3D cell reconstructions. When the cells are close packed, the contour grouping problem is more complex than that found in medical imaging, for example, because there are multiple regions of interest, the regions are not separable from each other by an identifiable background and regions cannot be distinguished by intensity differences. Here, we present an algorithm that uses three primary metrics-overlap of contour areas in adjacent images, co-linearity of the centroids of these areas across three images in a stack, and cell taper-to assign cells to groups. Decreasing thresholds are used to successively assign contours whose membership is less obvious. In a final step, remaining contours are assigned to existing groups by setting all thresholds to zero and groups having strong hour-glass shapes are partitioned. When applied to synthetic data from isotropic model aggregates, a curved model epithelium in which the long axes of the cells lie at all possible angles to the transection plane, and a confocal image stack, algorithm assignments were between 97 and 100% accurate in sets having at least four contours per cell. The algorithm is not particularly sensitive to the thresholds used, and a single set of parameters was used for all of the tests. The algorithm, which could be extended to time-lapse data, solves a key problem in the translation of image data into cell information.

  8. 3D differentiation of neural stem cells in macroporous photopolymerizable hydrogel scaffolds.

    Directory of Open Access Journals (Sweden)

    Hang Li

    Full Text Available Neural stem/progenitor cells (NSPCs are the stem cell of the adult central nervous system (CNS. These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes, thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm(2, 10 wt%, 6330±1160 µm(2, 20 wt%, 7600±1550 µm(2 compared with controls (0 wt%, 3150±220 µm(2. Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.

  9. SURVIVAL OF LIVER CELLS, IMMOBILIZED ON 3D-MATRIXES, IN LIVER FAILURE MODEL

    Directory of Open Access Journals (Sweden)

    M. Y. Shagidulin

    2011-01-01

    Full Text Available It was examined a new method for correction of hepatic failure by transplantation of liver support biounit (liver cells, immobilized on biocompatible and biodegradable 3D-matrixes ElastoPOB® into small intestine mesentery. It was determined that after modeling of acute hepatic failure on dogs by 65–70% liver resection and transplantation liver support biounit the restoration of disturbed biochemical indecies (such as total protein, lactate, cytolytic ensymes-ALT, AST, ALP, LDH, fibrinogen, protrombine index and others took place more rapidly on 9–14th day instead of 18th day in control. It was made a preposition about efficiency of the suggested method for correction both acute hepatic failure because even 90 days after transplantation of liver support biounit alive hepatocytes and neogenic plethoric vessels, growing through matrix were revealed. 

  10. Rearrangements of MYC gene facilitate risk stratification in diffuse large B-cell lymphoma patients treated with rituximab-CHOP

    DEFF Research Database (Denmark)

    Tzankov, Alexandar; Xu-Monette, Zijun Y; Gerhard, Marc;

    2014-01-01

    In order to address the debatable prognostic role of MYC rearrangements in diffuse large B-cell lymphoma patients treated with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, we evaluated MYC rearrangements by fluorescence in situ hybridization in 563 cases using br...

  11. Self-Organization of Polarized Cerebellar Tissue in 3D Culture of Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Keiko Muguruma

    2015-02-01

    Full Text Available During cerebellar development, the main portion of the cerebellar plate neuroepithelium gives birth to Purkinje cells and interneurons, whereas the rhombic lip, the germinal zone at its dorsal edge, generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components cooperate to form the intricate cerebellar structure. Here, we found that a polarized cerebellar structure self-organizes in 3D human embryonic stem cell (ESC culture. The self-organized neuroepithelium differentiates into electrophysiologically functional Purkinje cells. The addition of fibroblast growth factor 19 (FGF19 promotes spontaneous generation of dorsoventrally polarized neural-tube-like structures at the level of the cerebellum. Furthermore, addition of SDF1 and FGF19 promotes the generation of a continuous cerebellar plate neuroepithelium with rhombic-lip-like structure at one end and a three-layer cytoarchitecture similar to the embryonic cerebellum. Thus, human-ESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellum at the first trimester.

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

    Science.gov (United States)

    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.

  13. A 3D model for PEM fuel cells operated on reformate

    Science.gov (United States)

    Zhou, Tianhong; Liu, Hongtan

    A three-dimensional mathematical model for PEM fuel cells operated on reformate is developed based on our previous established fuel cell model [Int. J. Transport Phenomena 3 (2001) 177], by incorporating the adsorption and oxidation kinetics of CO on platinum surface proposed by Springer et al. [Proceedings of the Electrochemical Society, Montreal, Canada, 1997; J. Electrochem. Soc. 148 (2001) A11]. This model is capable of studying the effect of CO poisoning as well as the hydrogen dilution effect by inert gases. The adsorption and oxidation kinetics of CO on a platinum surface are incorporated in the source terms of the species equations; thus, the basic form of the mathematical equations are the same as those used for PEM fuel cells operated on pure hydrogen. With this model, we can obtain detailed information on the CO poisoning and variation of CO and hydrogen concentrations inside the anode. The results from this 3D model reveal many new phenomena that cannot be obtained from previous 1D or 2D models. Results of the effects of various operating and design parameters, such as anode flow rate, gas diffuser porosity, gas diffuser thickness, and the width of the collector plate shoulder, are also presented. The modeling results demonstrate the value of this model as a design and optimization tool for the anode of PEM fuel cells operating on reformate.

  14. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

    Science.gov (United States)

    Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

    2016-09-16

    3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2012-06-01

    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 × 25 × 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). Immunocytoche mistry 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 biochemical 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.

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

    Science.gov (United States)

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

    2016-09-06

    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.

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

    Science.gov (United States)

    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/cm2 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

  19. Generation of Multilayered 3D Structures of HepG2 Cells Using a Bio-printing Technique

    Science.gov (United States)

    Jeon, Hyeryeon; Kang, Kyojin; Park, Su A; Kim, Wan Doo; Paik, Seung Sam; Lee, Sang-Hun; Jeong, Jaemin; Choi, Dongho

    2017-01-01

    Background/Aims Chronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare. Methods A 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liver-specific markers was quantified on days 1, 7, 14, and 21. Results The cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture. Conclusions The 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver. PMID:27559001

  20. Clonal rearrangements of immunoglobulin genes and progression to B cell lymphoma in cutaneous lymphoid hyperplasia.

    Science.gov (United States)

    Wood, G S; Ngan, B Y; Tung, R; Hoffman, T E; Abel, E A; Hoppe, R T; Warnke, R A; Cleary, M L; Sklar, J

    1989-07-01

    Cutaneous lymphoid hyperplasia (CLH) is a disorder characterized by the development of one or more skin lesions containing dense lymphoid infiltrates that exhibit the histopathologic features of a benign, reactive process. Nevertheless, some cases have been associated with the subsequent development of clinically overt lymphomas. This suggests that monoclonal populations may exist in some cases of CLH and that these cases may represent a subset more likely to evolve into lymphoma. To determine if such a subset of CLH can be distinguished, Southern blot analysis of DNA was used to study the immunogenotypic features of lesions from 14 patients with clinical, histopathologic, and immunopathologic findings characteristic of CLH. Five cases exhibited detectable clonal rearrangements of immunoglobulin genes. Furthermore, one of these five cases evolved into overt diffuse large cell lymphoma of B cell lineage during a 2-year follow-up of recurrent disease at the original cutaneous site. The immunoglobulin gene rearrangements of this lymphoma were identical to those of the prior CLH lesion. There was no evidence of detectable t(14;18) chromosomal translocations or clonal rearrangements of the beta gene of the T cell receptor in any case. It was concluded that CLH can be divided into two subsets based on the presence or absence of a clonal B cell population, and that overt lymphoma can arise from the former subset and contain the same B cell clone identified in the pre-existent CLH lesion.

  1. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models.

    Science.gov (United States)

    Ricci, Claudio; Mota, Carlos; Moscato, Stefania; D'Alessandro, Delfo; Ugel, Stefano; Sartoris, Silvia; Bronte, Vincenzo; Boggi, Ugo; Campani, Daniela; Funel, Niccola; Moroni, Lorenzo; Danti, Serena

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

  2. Optimizing fuel cell parts by using 3D screen printed metals

    Energy Technology Data Exchange (ETDEWEB)

    Studnitzky, Thomas [Fraunhofer-Institue for Manufacturing and Advanced Materials, Dresden (Germany). Dept. of Powder Metallurgy and Composite Materials; Strauss, Alexander [Centre for Fuel Cell Technology, Duisburg (Germany). Dept. of Microsystems

    2010-07-01

    Miniature fuel cells have the potential to extend the runtime of various portable applications. In this context, sufficient energy densities have to be achieved within a stack in order to build fuel cell systems competitive to established battery technologies. Metallic bipolar plates composed of stainless steels permit the construction of thin and mechanically robust cells. Moreover the utilisation of the presented manufacturing method enables a new freedom in design of Bipolar Plates and their integrated flow field structures for future improvements of cell efficiency. In this study different miniature PEM fuel cells have been designed and tested. Bases for their construction are micro structured plates composed of 316L stainless steel. This design can include very fine walls down to 60 {mu}m as well as undercuts, which is impossible with other manufacturing methods. These designs were adapted by Fraunhofer IFAM for the screen printing process. As a first result a proof of concept has been established for 3D screen printing as a method for the manufacturing of fuel cell bipolar elements. To this end, a modified screen printing process is used to manufacture 3-dimensional parts layer-on-layer by depositing a suitable metallic powder which is mixed with a binder. The resulting green parts might include closed channels and channel wall thicknesses may reach 80 {mu}m or less. The green parts are debindered and sintered in order to obtain purely metallic structures. The manufactured bipolar plates show promising electric behaviour. In the current state of the project, bipolar plates with undercuts and new materials combinations are in the centre of the investigations. (orig.)

  3. 3D optical simulation formalism OPTOS for textured silicon solar cells.

    Science.gov (United States)

    Tucher, Nico; Eisenlohr, Johannes; Kiefel, Peter; Höhn, Oliver; Hauser, Hubert; Peters, Marius; Müller, Claas; Goldschmidt, Jan Christoph; Bläsi, Benedikt

    2015-11-30

    In this paper we introduce the three-dimensional formulation of the OPTOS formalism, a matrix-based method that allows for the efficient simulation of non-coherent light propagation and absorption in thick textured sheets. As application examples, we calculate the absorptance of solar cells featuring textures on front and rear side with different feature sizes operating in different optical regimes. A discretization of polar and azimuth angle enables a three-dimensional description of systems with arbitrary surface textures. We present redistribution matrices for 3D surface textures, including pyramidal textures, binary crossed gratings and a Lambertian scatterer. The results of the OPTOS simulations for silicon sheets with different combinations of these surfaces are in accordance with both optical measurements and results based on established simulation methods like ray tracing. Using OPTOS, we show that the integration of a diffractive grating at the rear side of a silicon solar cell featuring a pyramidal front side results in absorption close to the Yablonovitch Limit enhancing the photocurrent density by 0.6 mA/cm2 for a 200 µm thick cell.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

    Science.gov (United States)

    Kokovic, Ira; Jezersek Novakovic, Barbara; Novakovic, Srdjan

    2015-03-01

    Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

  6. Related pituitary cell lineages develop into interdigitated 3D cell networks.

    Science.gov (United States)

    Budry, Lionel; Lafont, Chrystel; El Yandouzi, Taoufik; Chauvet, Norbert; Conéjero, Geneviève; Drouin, Jacques; Mollard, Patrice

    2011-07-26

    The pituitary gland has long been considered to be a random patchwork of hormone-producing cells. By using pituitary-scale tridimensional imaging for two of the least abundant cell lineages, the corticotropes and gonadotropes, we have now uncovered highly organized and interdigitated cell networks that reflect homotypic and heterotypic interactions between cells. Although newly differentiated corticotrope cells appear on the ventral surface of the gland, they rapidly form homotypic strands of cells that extend from the lateral tips of the anterior pituitary along its ventral surface and into the medial gland. As the corticotrope network is established away from the microvasculature, cell morphology changes from rounded, to polygonal, and finally to cells with long cytoplasmic processes or cytonemes that connect corticotropes to the perivascular space. Gonadotropes differentiate later and are positioned in close proximity to corticotropes and capillaries. Blockade of corticotrope terminal differentiation produced by knockout of the gene encoding the transcription factor Tpit results in smaller gonadotropes within an expanded cell network, particularly in the lateral gland. Thus, pituitary-scale tridimensional imaging reveals highly structured cell networks of unique topology for each pituitary lineage. The sequential development of interdigitated cell networks during organogenesis indicate that extensive cell:cell interactions lead to a highly ordered cell positioning rather than random patchwork.

  7. Scaffolds for 3D in vitro culture of neural lineage cells.

    Science.gov (United States)

    Murphy, Ashley R; Laslett, Andrew; O'Brien, Carmel M; Cameron, Neil R

    2017-03-01

    Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions for this exciting and important field of biomaterials research.

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

    Energy Technology Data Exchange (ETDEWEB)

    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'.

  9. The Interactorium: visualising proteins, complexes and interaction networks in a virtual 3-D cell.

    Science.gov (United States)

    Widjaja, Yose Y; Pang, Chi Nam Ignatius; Li, Simone S; Wilkins, Marc R; Lambert, Tim D

    2009-12-01

    Here, we describe the Interactorium, a tool in which a Virtual Cell is used as the context for the seamless visualisation of the yeast protein interaction network, protein complexes and protein 3-D structures. The tool has been designed to display very complex networks of up to 40 000 proteins or 6000 multiprotein complexes and has a series of toolboxes and menus to allow real-time data manipulation and control the manner in which data are displayed. It incorporates new algorithms that reduce the complexity of the visualisation by the generation of putative new complexes from existing data and by the reduction of edges through the use of protein "twins" when they occur in multiple locations. Since the Interactorium permits multi-level viewing of the molecular biology of the cell, it is a considerable advance over existing approaches. We illustrate its use for Saccharomyces cerevisiae but note that it will also be useful for the analysis of data from simpler prokaryotes and higher eukaryotes, including humans. The Interactorium is available for download at http://www.interactorium.net.

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

    NARCIS (Netherlands)

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

    2016-01-01

    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 doma

  11. The performance of 3-D graphite doped anodes in microbial electrolysis cells

    Science.gov (United States)

    Yasri, Nael G.; Nakhla, George

    2017-02-01

    This study investigated the use of granular activated carbon (GAC) as high surface area 3-dimensional (3-D) anode in MECs systems. The interfacial anodes' charge transfer resistance of the doped GAC did not impact the overall performance of MECs. Based on our finding, the 3-D anode packed with GAC-doped with nonconductive calcium sulfide (CaS) outperformed the more conductive iron (II) sulfide (FeS), magnetite (Fe3O4), or GAC without doping. The results showed higher current densities for 3-D CaS (40.1 A/m3), as compared with 3-D FeS (34.4 A/m3), 3-D Fe3O4 (29.8 A/m3), and 3-D GAC (23.1 A/m3). The higher current density in the 3-D CaS translated to higher coulombic efficiency (96.7%), hydrogen yield (3.6 mol H2/mol acetate), and attached biomass per anode mass (54.01 mg COD biomass/g GAC). Although the 3-D MEC achieved similar hydrogen yield, hydrogen recovery efficiency, and COD removal rate to a conventional sandwich type MEC, the current density, coulombic efficiency, and overall energy efficiency were higher.

  12. A tetraphenylethylene core-based 3D structure small molecular acceptor enabling efficient non-fullerene organic solar cells.

    Science.gov (United States)

    Liu, Yuhang; Mu, Cheng; Jiang, Kui; Zhao, Jingbo; Li, Yunke; Zhang, Lu; Li, Zhengke; Lai, Joshua Yuk Lin; Hu, Huawei; Ma, Tingxuan; Hu, Rongrong; Yu, Demei; Huang, Xuhui; Tang, Ben Zhong; Yan, He

    2015-02-01

    A tetraphenylethylene core-based small molecular acceptor with a unique 3D molecular structure is developed. Bulk-heterojunction blend films with a small feature size (≈20 nm) are obtained, which lead to non-fullerene organic solar cells (OSCs) with 5.5% power conversion efficiency. The work provides a new molecular design approach to efficient non-fullerene OSCs based on 3D-structured small-molecule acceptors.

  13. Cells in 3D matrices under interstitial flow: effects of extracellular matrix alignment on cell shear stress and drag forces.

    Science.gov (United States)

    Pedersen, John A; Lichter, Seth; Swartz, Melody A

    2010-03-22

    Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman-Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.

  14. Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.

    Directory of Open Access Journals (Sweden)

    Michael S Weiss

    Full Text Available BACKGROUND: Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture. METHODOLOGY/PRINCIPAL FINDINGS: TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A. CONCLUSIONS/SIGNIFICANCE: This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a

  15. Melanoma cells undergo aggressive coalescence in a 3D Matrigel model that is repressed by anti-CD44

    Science.gov (United States)

    Voss, Edward; Kuhl, Spencer; Buchele, Emma C.; Klemme, Michael R.; Russell, Kanoe B.; Ambrose, Joseph; Soll, Benjamin A.; Bossler, Aaron; Milhem, Mohammed; Goldman, Charles

    2017-01-01

    Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid. Non-tumorigenic cells did not undergo coalescence. Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model. Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model. Normal melanocytes did not. However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic. A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence. They also blocked coalescence of tumorigenic cells derived from a breast tumor. These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs. PMID:28264026

  16. Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells.

    Science.gov (United States)

    Hara, Jared; Tottori, Jordan; Anders, Megan; Dadhwal, Smritee; Asuri, Prashanth; Mobed-Miremadi, Maryam

    2017-05-01

    Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L9 (3(4)) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L9 study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.

  17. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix.

    Science.gov (United States)

    Ma, Yufei; Ji, Yuan; Huang, Guoyou; Ling, Kai; Zhang, Xiaohui; Xu, Feng

    2015-12-22

    Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue.

  18. A Bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors.

    Science.gov (United States)

    Bouyer, Charlène; Chen, Pu; Güven, Sinan; Demirtaş, Tuğrul Tolga; Nieland, Thomas J F; Padilla, Frédéric; Demirci, Utkan

    2016-01-06

    A bio-acoustic levitational assembly method for engineering of multilayered, 3D brainlike constructs is presented. Acoustic radiation forces are used to levitate neuroprogenitors derived from human embryonic stem cells in 3D multilayered fibrin tissue constructs. The neuro-progenitor cells are subsequently differentiated in neural cells, resulting in a 3D neuronal construct with inter and intralayer neurite elongations.

  19. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

    Science.gov (United States)

    Moreno, Edinson Lucumi; Hachi, Siham; Hemmer, Kathrin; Trietsch, Sebastiaan J; Baumuratov, Aidos S; Hankemeier, Thomas; Vulto, Paul; Schwamborn, Jens C; Fleming, Ronan M T

    2015-06-07

    A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

  20. Highly ordered large-scale neuronal networks of individual cells - toward single cell to 3D nanowire intracellular interfaces.

    Science.gov (United States)

    Kwiat, Moria; Elnathan, Roey; Pevzner, Alexander; Peretz, Asher; Barak, Boaz; Peretz, Hagit; Ducobni, Tamir; Stein, Daniel; Mittelman, Leonid; Ashery, Uri; Patolsky, Fernando

    2012-07-25

    The use of artificial, prepatterned neuronal networks in vitro is a promising approach for studying the development and dynamics of small neural systems in order to understand the basic functionality of neurons and later on of the brain. The present work presents a high fidelity and robust procedure for controlling neuronal growth on substrates such as silicon wafers and glass, enabling us to obtain mature and durable neural networks of individual cells at designed geometries. It offers several advantages compared to other related techniques that have been reported in recent years mainly because of its high yield and reproducibility. The procedure is based on surface chemistry that allows the formation of functional, tailormade neural architectures with a micrometer high-resolution partition, that has the ability to promote or repel cells attachment. The main achievements of this work are deemed to be the creation of a large scale neuronal network at low density down to individual cells, that develop intact typical neurites and synapses without any glia-supportive cells straight from the plating stage and with a relatively long term survival rate, up to 4 weeks. An important application of this method is its use on 3D nanopillars and 3D nanowire-device arrays, enabling not only the cell bodies, but also their neurites to be positioned directly on electrical devices and grow with registration to the recording elements underneath.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Recent advance in fabricating monolithic 3D porous graphene and their applications in biosensing and biofuel cells.

    Science.gov (United States)

    Qiu, Hua-Jun; Guan, Yongxin; Luo, Pan; Wang, Yu

    2017-03-15

    Graphene shows great potential in biosensing and bioelectronics. To facilitate graphene's applications and enhance its performance, recently, three-dimensional (3D) graphene-based materials especially free-standing porous graphene with tunable pore size and void space, have attracted increasing attention for bio-related applications owing to their special features. 3D graphene usually shows the following merits such as an interconnected porous network, a high electronic conductivity, a large active surface area, good chemical/thermal stability and can be more easily handled compared with dispersed graphene sheets. With modified surface properties, graphene can also be bio-friendly. These properties make 3D graphene a perfect candidate as high-performance electrode materials in bioelectronics devices. In this review, we discuss recent advance in fabricating monolithic 3D graphene and their applications in biosensing and biofuel cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    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)

  4. An impedance method for spatial sensing of 3D cell constructs – towards applications in tissue engineering

    DEFF Research Database (Denmark)

    Canali, Chiara; Mazzoni, Chiara; Larsen, Layla Bashir

    2015-01-01

    ) cells were encapsulated in gelatin to form artificial 3D cell constructs and detected when placed in different positions inside large gelatin scaffolds. Taken together, these results open new perspectives for impedance-based sensing technologies for non-invasive monitoring in tissue engineering...

  5. A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

    Directory of Open Access Journals (Sweden)

    Robert J Polackwich

    Full Text Available New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1 increased the strength of cell-induced forces on the ECM, 2 did not significantly change migration speed, and 3 increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

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

    Science.gov (United States)

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

    2017-01-27

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

  7. A multimaterial bioink method for 3D printing tunable, cell-compatible hydrogels.

    Science.gov (United States)

    Rutz, Alexandra L; Hyland, Kelly E; Jakus, Adam E; Burghardt, Wesley R; Shah, Ramille N

    2015-03-04

    A multimaterial bio-ink method using polyethylene glycol crosslinking is presented for expanding the biomaterial palette required for 3D bioprinting of more mimetic and customizable tissue and organ constructs. Lightly crosslinked, soft hydrogels are produced from precursor solutions of various materials and 3D printed. Rheological and biological characterizations are presented, and the promise of this new bio-ink synthesis strategy is discussed.

  8. Pulmonary surfactant expression analysis--role of cell-cell interactions and 3-D tissue-like architecture.

    Science.gov (United States)

    Nandkumar, Maya A; Ashna, U; Thomas, Lynda V; Nair, Prabha D

    2015-03-01

    Surfactant production is important in maintaining alveolar function both in vivo and in vitro, but surfactant expression is the primary property lost by alveolar Type II Pneumocytes in culture and its maintenance is a functional requirement. To develop a functional tissue-like model, the in vivo cell-cell interactions and three dimensional architecture has to be reproduced. To this end, 3D button-shaped synthetic gelatin vinyl acetate (GeVAc) co-polymer scaffold was seeded with different types of lung cells. Functionality of the construct was studied under both static and dynamic conditions. The construct was characterized by Environmental Scanning Electron and fluorescent microscopy, and functionality of the system was analyzed by studying mRNA modulations of all four surfactant genes A, B, C, and D by real time-PCR and varying culture conditions. The scaffold supports alveolar cell adhesion and maintenance of cuboidal morphology, and the alveolar-specific property of surfactant synthesis, which would otherwise be rapidly lost in culture. This is a novel 3D system that expresses all 4 surfactants for a culture duration of 3 weeks.

  9. Deletional rearrangement in the human T-cell receptor. cap alpha. -chain locus

    Energy Technology Data Exchange (ETDEWEB)

    de Villartay, J.P.; Lewis, D.; Hockett, R.; Waldmann, T.A.; Korsmeyer, S.J.; Cohen, D.I.

    1987-12-01

    The antigen-specific receptor on the surface of mature T lymphocytes is a heterodimer consisting of polypeptides termed ..cap alpha.. and ..beta... In the course of characterizing human T-cell tumors with an immature (CD4/sup -/, CD8/sup -/) surface phenotype, the authors detected a 2-kilobase ..cap alpha..-related transcript. Analysis of cDNA clones corresponding to this transcript established that a genetic element (which they call TEA, for T early ..cap alpha..) located between the ..cap alpha..-chain variable- and joining-region genes had been spliced to the ..cap alpha.. constant region. The TEA transcript is present early in thymocyte ontogeny, and its expression declines during T-cell maturation. More important, the TEA area functions as an active site for rearrangement within the ..cap alpha.. gene locus. Blot hybridization of restriction enzyme-digested DNA with a TEA probe revealed a narrowly limited pattern of rearrangement in polyclonal thymic DNA, surprisingly different from the pattern expected for the mature ..cap alpha.. gene with its complex diversity. These DNA blots also showed that TEA is generally present in the germ-line configuration in cells expressing the ..gamma..delta heterodimeric receptor and is deleted from mature (..cap alpha beta..-expressing) T-lymphocyte tumors and lines. Moreover, the TEA transcript lacked a long open reading frame for protein but instead possessed multiple copies of a repetitive element resembling those utilized in the heavy-chain class switch of the immunoglobulin genes. The temporal nature of the rearrangements and expression detected by TEA suggests that this recombination could mediate a transition between immature (..gamma..delta-expressing) T cells and mature (..cap alpha beta..-expressing) T cells.

  10. Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE

    Science.gov (United States)

    Carmona, Guillaume; Perera, Upamali; Gillett, Cheryl; Naba, Alexandra; Law, Ah-Lai; Sharma, Ved P.; Wang, Jian; Wyckoff, Jeffrey; Balsamo, Michele; Mosis, Fuad; De Piano, Mario; Monypenny, James; Woodman, Natalie; McConnell, Russell E.; Mouneimne, Ghassan; Van Hemelrijck, Mieke; Cao, Yihai; Condeelis, John; Hynes, Richard O.; Gertler, Frank B.; Krause, Matthias

    2016-01-01

    Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlates with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation, and matrix degradation were impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not Ena/VASP is required for random 2D cell migration. We identify a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, while Src-dependent phosphorylation enhances binding to Scar/WAVE but not Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of EGF gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis. PMID:26996666

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    Soto Ana M

    2010-06-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusions 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

  13. Quantifying transient 3D dynamical phenomena of single mRNA particles in live yeast cell measurements.

    Science.gov (United States)

    Calderon, Christopher P; Thompson, Michael A; Casolari, Jason M; Paffenroth, Randy C; Moerner, W E

    2013-12-12

    Single-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4 ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions. There is a great need for new analysis tools aiming to more fully extract the biological information content from in vivo SPT measurements. High-resolution SPT experimental data has enormous potential to objectively scrutinize various proposed mechanistic schemes arising from theoretical biophysics and cell biology. At the same time, methods for rigorously checking the statistical consistency of both model assumptions and estimated parameters against observed experimental data (i.e., goodness-of-fit tests) have not received great attention. We demonstrate methods enabling (1) estimation of the parameters of 3D stochastic differential equation (SDE) models of the underlying dynamics given only one trajectory; and (2) construction of hypothesis tests checking the consistency of the fitted model with the observed trajectory so that extracted parameters are not overinterpreted (the tools are applicable to linear or nonlinear SDEs calibrated from nonstationary time series data). The approach is demonstrated on high-resolution 3D trajectories of single ARG3 mRNA particles in yeast cells in order to show the power of the methods in detecting signatures of transient directed transport. The methods presented are generally relevant to a wide variety of 2D and 3D SPT

  14. Sustained PDGF-BB release from PHBHHx loaded nanoparticles in 3D hydrogel/stem cell model.

    Science.gov (United States)

    Dong, Cui-Ling; Webb, William R; Peng, Qiang; Tang, James Z; Forsyth, Nicholas R; Chen, Guo-Qiang; El Haj, Alicia J

    2015-01-01

    This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    Science.gov (United States)

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  17. Preparation of 3D electrode microarrays of multi-walled carbon nanotubes/nafion nanocomposites for microfluidic biofuel cells.

    Science.gov (United States)

    Choi, Jin Ho; Kim, Young Ho; Choi, Sung Deuk; Kim, Gyu Man

    2014-12-01

    Three-dimensional (3D) electrode microarrays with multi-walled carbon nanotubes (MWCNTs) reinforced Nafion nanocomposites were prepared for microfluidic biofuel cells. The oxidized MWCNTs (ox-MWCNTs) were prepared using chemical reactions with 60% nitric acid solution with pristine MWCNTs at 120 degrees C for 12 hrs with a nitrogen gas flow environment. Ox-MWCNTs in the range of 1 to 20 wt.% based on the Nafion polymer weight were reinforced to Nafion nanocomposites by solution casting. The micro-porous structure of the ox-MWCNTs reinforced Nafion nanocomposites was prepared by plasma etching for 5 to 20 min. The 10 wt.% ox-MWCNTs reinforced Nafion nanocomposite produced stable micro-porous structures of 3D electrodes by 10 min plasma etching. Micro-scale 3D structures of MWCNTs reinforced Nafion nanocomposites in a diameter range of 47 to 300 μm were prepared by the micro-stencil assisted casting. To characterize the 3D electrode microarrays, the physical geometry and the reinforced MWCNT dispersion in the nanocomposite structure were examined using a scanning electron microscope (SEM) and an optical microscope. Thermal property measurements of the ox-MWCNTs reinforced Nafion nanocomposites with 10 min of plasma etching, and without plasma etching were made. Both showed stable thermal properties over 300 degrees C. The proposed 3D electrode microarray of MWCNT/Nafion nanocomposites with micro-porous structures can be applied to miniaturized fuel cell devices.

  18. Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Anna C Luca

    Full Text Available Three-dimensional (3D tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC cell lines. LrECM on-top (3D culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular

  19. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    Energy Technology Data Exchange (ETDEWEB)

    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

  20. Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

    OpenAIRE

    2007-01-01

    The absence of Tsa1, a key peroxiredoxin that functions to scavenge H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the ...

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

    DEFF Research Database (Denmark)

    Fey, S. J.; Wrzesinski, Krzysztof

    2012-01-01

    , diclofenac, metformin, phenformin, and valproic acid) to LD50 data (mg compound/mg cellular protein) showed that the variation in LD50 values was generally less than that suggested by the original LC50 data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented...

  2. Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.

    Science.gov (United States)

    Meng, Xiaoting; Li, Wenfei; Young, Fraser; Gao, Runchi; Chalmers, Laura; Zhao, Min; Song, Bing

    2012-02-16

    Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system(1,2). These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans(3,4). In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types(5,6), including neural progenitor cells (NPCs)(7,8). Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously(5,11). Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures(9,10). Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

  3. Cytoskeleton rearrangement induced by tetraspanin engagement modulates the activation of T and NK cells.

    Science.gov (United States)

    Crotta, Stefania; Ronconi, Vanessa; Ulivieri, Cristina; Baldari, Cosima T; Valiante, Nicholas M; Valiente, Nicholas M; Abrignani, Sergio; Wack, Andreas

    2006-04-01

    The hepatitis C virus (HCV) binds to human cells through the interaction of its envelope glycoprotein E2 with the tetraspanin CD81. We have previously reported that engagement of CD81 has opposite effects on T and NK cell function, as it enhances T cell receptor-mediated T cell activation and inhibits CD16- or IL-12-mediated NK cell activation. We further investigated this dichotomy and found that another tetraspanin, CD82, induces the same opposing effects on human primary T and NK cells. Activation by other unrelated stimuli such as NKG2D- and beta-1 integrin is also reduced by CD81 ligation on NK cells. CD81 engagement by monoclonal antibody or HCV-E2 enhances zeta and Erk phosphorylation in T cells and reduces them in NK cells, reflecting the opposite functional outcomes. CD81 engagement induces dramatic morphological changes and local F-actin accumulation in both NK and T cells, indicating rearrangement of the actin cytoskeleton. Pharmacological inhibition of actin polymerization reduces T cell activation, whereas it greatly enhances NK cell activation. Importantly, treatment with actin blockers abolishes the inhibitory effect of CD81 ligation on NK cells. We propose that tetraspanin engagement leads to comparable cytoskeleton reorganization in T and NK cells, which in turn results in opposite functional outcomes.

  4. Enabling Lorentz boosted frame particle-in-cell simulations of laser wakefield acceleration in quasi-3D geometry

    Science.gov (United States)

    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 ∼ γb2 = (1 - βb2)-1. Meanwhile, when LWFA is modeled in the quasi-3D geometry in which the electromagnetic fields and current are decomposed into a limited number of azimuthal harmonics, speedups are achieved by modeling three dimensional (3D) problems with the computational loads on the order of two dimensional r - z simulations. Here, we describe a method to combine the speedups from the Lorentz boosted frame and quasi-3D algorithms. The key to the combination is the use of a hybrid Yee-FFT solver in the quasi-3D geometry that significantly mitigates the Numerical Cerenkov Instability (NCI) which inevitably arises in a Lorentz boosted frame due to the unphysical coupling of Langmuir modes and EM modes of the relativistically drifting plasma in these simulations. In addition, based on the 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.

  5. Advanced 3D Poisson solvers and particle-in-cell methods for accelerator modeling

    Energy Technology Data Exchange (ETDEWEB)

    Serafini, David B; McCorquodale, Peter; Colella, Phillip [Lawrence Berkeley National Lab, Applied Numerical Algorithms Group, SciDAC Applied Differential Equations Center (United States)

    2005-01-01

    We seek to improve on the conventional FFT-based algorithms for solving the Poisson equation with infinite-domain (open) boundary conditions for large problems in accelerator modeling and related areas. In particular, improvements in both accuracy and performance are possible by combining several technologies: the method of local corrections (MLC); the James algorithm; and adaptive mesh refinement (AMR). The MLC enables the parallelization (by domain decomposition) of problems with large domains and many grid points. This improves on the FFT-based Poisson solvers typically used as it doesn't require the all-to-all communication pattern that parallel 3d FFT algorithms require, which tends to be a performance bottleneck on current (and foreseeable) parallel computers. In initial tests, good scalability up to 1000 processors has been demonstrated for our new MLC solver. An essential component of our approach is a new version of the James algorithm for infinite-domain boundary conditions for the case of three dimensions. By using a simplified version of the fast multipole method in the boundary-to-boundary potential calculation, we improve on the performance of the Hockney algorithm typically used by reducing the number of grid points by a factor of 8, and the CPU costs by a factor of 3. This is particularly important for large problems where computer memory limits are a consideration. The MLC allows for the use of adaptive mesh refinement, which reduces the number of grid points and increases the accuracy in the Poisson solution. This improves on the uniform grid methods typically used in PIC codes, particularly in beam problems where the halo is large. Also, the number of particles per cell can be controlled more closely with adaptivity than with a uniform grid. To use AMR with particles is more complicated than using uniform grids. It affects depositing particles on the non-uniform grid, reassigning particles when the adaptive grid changes and maintaining the

  6. Extensive junctional diversity of rearranged human T cell receptor delta genes.

    Science.gov (United States)

    Hata, S; Satyanarayana, K; Devlin, P; Band, H; McLean, J; Strominger, J L; Brenner, M B; Krangel, M S

    1988-06-10

    The human T cell receptor delta (TCR delta) gene encodes one component of the TCR gamma delta-CD3 complex found on subsets of peripheral blood and thymic T cells. Human TCR delta diversity was estimated by characterizing rearrangements in TCR gamma delta cell lines and determining the structures of complementary DNA clones representing functional and nonfunctional transcripts in these cell lines. One V delta segment and one J delta segment were identified in all functional transcripts, although a distinct J delta segment was identified in a truncated transcript. Further, one D delta element was identified, and evidence for the use of an additional D delta element was obtained. Thus human TCR delta genes appear to use a limited number of germline elements. However, the apparent use of two D delta elements in tandem coupled with imprecise joining and extensive incorporation of N nucleotides generates unprecedented variability in the junctional region.

  7. Analyzing the CDR3 Repertoire with respect to TCR-Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS.

    Science.gov (United States)

    Ma, Long; Yang, Liwen; Bin Shi; He, Xiaoyan; Peng, Aihua; Li, Yuehong; Zhang, Teng; Sun, Suhong; Ma, Rui; Yao, Xinsheng

    2016-07-12

    V-D-J rearrangement of the TCR-beta chain follows the 12/23 rule and the beyond 12/23 restriction. Currently, the proportion and characteristics of TCR-beta chain V-J rearrangement is unclear. We used high-throughput sequencing to compare and analyze TCR-beta chain V-J rearrangement and V-D-J rearrangement in the CDR3 repertoires of T cells from the PBMCs of six volunteers and six BALB/c mice. The results showed that the percentage of V-J rearrangement of the volunteers was approximately 0.7%, whereas that of the mice was 2.2%. The clonality of mice V-J rearrangement was significantly reduced compared with the V-D-J rearrangement, whereas the clonality of human V-J rearrangement was slightly reduced compared with the V-D-J rearrangement. V-J rearrangement in CDR3 involved the significant usage of N, S, F and L, whereas V-D-J rearrangement in CDR3 involved the significant usage of R and G. The levels of V deletion and J deletion in V-J rearrangement were significantly reduced compared with V-D-J rearrangement. TRBD and TRBJ usage in V-J rearrangement differed from that of V-D-J rearrangement, including dominant usage of TRBV and TRBJ and their pairing. Taken together, these results provide new ideas and technology for studies of V-D-J rearrangement and V-J rearrangement in the CDR3 repertoire.

  8. 3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair.

    Science.gov (United States)

    Hsieh, Fu-Yu; Lin, Hsin-Hua; Hsu, Shan-Hui

    2015-12-01

    The 3D bioprinting technology serves as a powerful tool for building tissue in the field of tissue engineering. Traditional 3D printing methods involve the use of heat, toxic organic solvents, or toxic photoinitiators for fabrication of synthetic scaffolds. In this study, two thermoresponsive water-based biodegradable polyurethane dispersions (PU1 and PU2) were synthesized which may form gel near 37 °C without any crosslinker. The stiffness of the hydrogel could be easily fine-tuned by the solid content of the dispersion. Neural stem cells (NSCs) were embedded into the polyurethane dispersions before gelation. The dispersions containing NSCs were subsequently printed and maintained at 37 °C. The NSCs in 25-30% PU2 hydrogels (∼680-2400 Pa) had excellent proliferation and differentiation but not in 25-30% PU1 hydrogels. Moreover, NSC-laden 25-30% PU2 hydrogels injected into the zebrafish embryo neural injury model could rescue the function of impaired nervous system. However, NSC-laden 25-30% PU1 hydrogels only showed a minor repair effect in the zebrafish model. In addition, the function of adult zebrafish with traumatic brain injury was rescued after implantation of the 3D-printed NSC-laden 25% PU2 constructs. Therefore, the newly developed 3D bioprinting technique involving NSCs embedded in the thermoresponsive biodegradable polyurethane ink offers new possibilities for future applications of 3D bioprinting in neural tissue engineering.

  9. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

    Science.gov (United States)

    Billiet, Thomas; Gevaert, Elien; De Schryver, Thomas; Cornelissen, Maria; Dubruel, Peter

    2014-01-01

    In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed.

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

    Science.gov (United States)

    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 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

    Science.gov (United States)

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

    2014-04-15

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  14. Role of nanotopography in the development of tissue engineered 3D organs and tissues using mesenchymal stem cells.

    Science.gov (United States)

    Salmasi, Shima; Kalaskar, Deepak M; Yoon, Wai-Weng; Blunn, Gordon W; Seifalian, Alexander M

    2015-03-26

    Recent regenerative medicine and tissue engineering strategies (using cells, scaffolds, medical devices and gene therapy) have led to fascinating progress of translation of basic research towards clinical applications. In the past decade, great deal of research has focused on developing various three dimensional (3D) organs, such as bone, skin, liver, kidney and ear, using such strategies in order to replace or regenerate damaged organs for the purpose of maintaining or restoring organs' functions that may have been lost due to aging, accident or disease. The surface properties of a material or a device are key aspects in determining the success of the implant in biomedicine, as the majority of biological reactions in human body occur on surfaces or interfaces. Furthermore, it has been established in the literature that cell adhesion and proliferation are, to a great extent, influenced by the micro- and nano-surface characteristics of biomaterials and devices. In addition, it has been shown that the functions of stem cells, mesenchymal stem cells in particular, could be regulated through physical interaction with specific nanotopographical cues. Therefore, guided stem cell proliferation, differentiation and function are of great importance in the regeneration of 3D tissues and organs using tissue engineering strategies. This review will provide an update on the impact of nanotopography on mesenchymal stem cells for the purpose of developing laboratory-based 3D organs and tissues, as well as the most recent research and case studies on this topic.

  15. 3D Plant Cell Architecture of Arabidopsis thaliana (Brassicaceae Using Focused Ion Beam–Scanning Electron Microscopy

    Directory of Open Access Journals (Sweden)

    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.

  16. The fabrication of double layer tubular vascular tissue engineering scaffold via coaxial electrospinning and its 3D cell coculture.

    Science.gov (United States)

    Ye, Lin; Cao, Jie; Chen, Lamei; Geng, Xue; Zhang, Ai-Ying; Guo, Lian-Rui; Gu, Yong-Quan; Feng, Zeng-Guo

    2015-12-01

    A continuous electrospinning technique was applied to fabricate double layer tubular tissue engineering vascular graft (TEVG) scaffold. The luminal layer was made from poly(ɛ-caprolac-tone)(PCL) ultrafine fibers via common single axial electrospinning followed by the outer layer of core-shell structured nanofibers via coaxial electrospinning. For preparing the outer layernano-fibers, the PCL was electrospun into the shell and both bovine serum albumin (BSA) and tetrapeptide val-gal-pro-gly (VAPG) were encapsulated into the core. The core-shell structure in the outer layer fibers was observed by transmission electron microscope (TEM). The in vitro release tests exhibited the sustainable release behavior of BSA and VAPG so that they provided a better cell growth environment in the interior of tubular scaffold wall. The in vitro culture of smooth muscle cells (SMCs) demonstrated their potential to penetrate into the scaffold wall for the 3D cell culture. Subsequently, 3D cell coculture was conducted. First, SMCs were seeded on the luminal surface of the scaffold and cultured for 5 days, and then endothelial cells (ECs) were also seeded on the luminal surface and cocultured with SMCs for another 2 days. After stained with antibodies, 3D cell distribution on the scaffold was revealed by confocal laser scanning microscopy (CLSM) where ECs were mainly located on the luminal surface whereas SMCs penetrated into the surface and distributed inside the scaffold wall. This double layer tubular scaffold with 3D cell distribution showed the promise to develop it into a novel TEVG for clinical trials in the near future.

  17. Laser 3D printing with sub-microscale resolution of porous elastomeric scaffolds for supporting human bone stem cells.

    Science.gov (United States)

    Petrochenko, Peter E; Torgersen, Jan; Gruber, Peter; Hicks, Lucas A; Zheng, Jiwen; Kumar, Girish; Narayan, Roger J; Goering, Peter L; Liska, Robert; Stampfl, Jürgen; Ovsianikov, Aleksandr

    2015-04-01

    A reproducible method is needed to fabricate 3D scaffold constructs that results in periodic and uniform structures with precise control at sub-micrometer and micrometer length scales. In this study, fabrication of scaffolds by two-photon polymerization (2PP) of a biodegradable urethane and acrylate-based photoelastomer is demonstrated. This material supports 2PP processing with sub-micrometer spatial resolution. The high photoreactivity of the biophotoelastomer permits 2PP processing at a scanning speed of 1000 mm s(-1), facilitating rapid fabrication of relatively large structures (>5 mm(3)). These structures are custom printed for in vitro assay screening in 96-well plates and are sufficiently flexible to enable facile handling and transplantation. These results indicate that stable scaffolds with porosities of greater than 60% can be produced using 2PP. Human bone marrow stromal cells grown on 3D scaffolds exhibit increased growth and proliferation compared to smooth 2D scaffold controls. 3D scaffolds adsorb larger amounts of protein than smooth 2D scaffolds due to their larger surface area; the scaffolds also allow cells to attach in multiple planes and to completely infiltrate the porous scaffolds. The flexible photoelastomer material is biocompatible in vitro and is associated with facile handling, making it a viable candidate for further study of complex 3D-printed scaffolds.

  18. REARRANGEMENT AND EXPRESSION OF T CELL RECEPTOR β GENE IN HUMAN HEMOPOIETIC CELL LINES AND PRIMARY CELLS FROM ACUTE LYMPHOCYTIC LEUKEMIAS

    Institute of Scientific and Technical Information of China (English)

    仇一华; 陈诗书

    1992-01-01

    Using Southern blot, Northern blot and Quick blot methods, we examined the rearrangement and expression of TCR βgene in four early differentiation stage cell lines from human hemopoietic system, namely HL-60, Jurkat, Daudi and Raji cells as well as lymphocytes from 17 acute lymphocytic leukemia (ALL) patients. The results showed. Ⅰ) Rearrangement of TCR βgene was seen in Jurkat cells. A germline pattern was observed in HL-60, Daudi and Raji cells. 2) Eight of 9 patients with T-ALL had cells with rearranged TCR βgene. But two of 3 patients with B-ALL and three of 5 patients with nonT, nonB-ALL also had cells with rearranged TCR βgene. 3) A 1.3 kb full-length transcript and a 1.0 kb truncated transcript were detected in Jurkat cells by probing with 32P-TCR βcDNA. But some leukemic B cells also expressed an incompleted transcript. 4) TCR βmRNA was detected in six of 8 patients with T-ALL, four of 5 patients with nonT, nonB-ALL and one of 3 patients with B-ALL. But the level of expression was quite differ ent. The dual-rearrangement and the abnormal expression may give us a new clue for researching leukemogenesis.

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

    Science.gov (United States)

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

    2016-03-07

    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.

  20. A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production

    Directory of Open Access Journals (Sweden)

    Tomomi G. Otsuji

    2014-05-01

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

  1. Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE.

    Science.gov (United States)

    Carmona, G; Perera, U; Gillett, C; Naba, A; Law, A-L; Sharma, V P; Wang, J; Wyckoff, J; Balsamo, M; Mosis, F; De Piano, M; Monypenny, J; Woodman, N; McConnell, R E; Mouneimne, G; Van Hemelrijck, M; Cao, Y; Condeelis, J; Hynes, R O; Gertler, F B; Krause, M

    2016-09-29

    Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.

  2. Rearrangements of chicken immunoglobulin genes in lymphoid cells transformed by the avian retroviral oncogene v-rel.

    Science.gov (United States)

    Chen, L; Lim, M Y; Bose, H; Bishop, J M

    1988-01-01

    The retroviral oncogene v-rel transforms poorly characterized lymphoid cells. We have explored the nature of these cells by analyzing the configuration and expression of immunoglobulin genes in chicken hemopoietic cells transformed by v-rel. None of the transformed cells expressed their immunoglobulin genes. The cells fell into three classes: class I cells have their immunoglobulin genes potentially in an embryonic configuration; class II and class III cells have lost one copy of the lambda light chain locus and have one copy of the heavy chain locus rearranged into a configuration that differs from what is found in mature B cells. In class II cells, the other heavy chain locus may be in embryonic configuration, whereas it is deleted in class III cells. The first of these classes may represent the earliest stage of the lymphoid lineage yet encountered among virus-transformed cells, whereas the second and third classes represent an apparently anomalous rearrangement whose origin remains unknown.

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

    Science.gov (United States)

    Knight, Eleanor; Przyborski, Stefan

    2015-12-01

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

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

    DEFF Research Database (Denmark)

    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...... 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...

  5. Synthesis and Characterization of a Photoelectrode with a Novel 3D Structure for Dye-Sensitized Solar Cells

    Directory of Open Access Journals (Sweden)

    Kun-Ching Cho

    2014-01-01

    Full Text Available This study designs a novel dye-sensitized solar cell (DSSC in which the photoanode is derived from its three-dimensional (3D structure. The inside of the cell has a positive illumination structure, with the purposes of increasing the area of photoelectrode thin film and of increasing the illuminated area within a fixed area in order to achieve the objective of enhancing the photoelectric conversion efficiency of cell. For the cell structure experiment, the study uses graphite paper, carbon and platinum as counter electrode materials, and then conducts measurement with cell heights of 3 mm, 5 mm, and 7 mm. The electrolyte used is a gel polymer electrolyte. The assembly of the cell is divided into vertical assembly, inclined assembly, and tandem assembly. In the 3D tandem cell experiment, the counter electrode material is platinum. Experimental results show that when cell height is 7 mm and illuminated area is 0.28 cm2, open-loop voltage is 0.662 V, short-circuit current density is 18.42 mA/cm2, fill factor is 0.31, and the photoelectric conversion efficiency is 3.85%, which is 1.65 times that under vertical assembly (2.34% and 2.15 times that of the flat cell (1.79%.

  6. Nanoengineered implant as a new platform for regenerative nanomedicine using 3D well-organized human cell spheroids

    Science.gov (United States)

    Keller, Laetitia; Idoux-Gillet, Ysia; Wagner, Quentin; Eap, Sandy; Brasse, David; Schwinté, Pascale; Arruebo, Manuel; Benkirane-Jessel, Nadia

    2017-01-01

    In tissue engineering, it is still rare today to see clinically transferable strategies for tissue-engineered graft production that conclusively offer better tissue regeneration than the already existing technologies, decreased recovery times, and less risk of complications. Here a novel tissue-engineering concept is presented for the production of living bone implants combining 1) a nanofibrous and microporous implant as cell colonization matrix and 2) 3D bone cell spheroids. This combination, double 3D implants, shows clinical relevant thicknesses for the treatment of an early stage of bone lesions before the need of bone substitutes. The strategy presented here shows a complete closure of a defect in nude mice calvaria after only 31 days. As a novel strategy for bone regenerative nanomedicine, it holds great promises to enhance the therapeutic efficacy of living bone implants. PMID:28138241

  7. 3D exploration of light scattering from live cells in the presence of gold nanomarkers using holographic microscopy

    CERN Document Server

    Joud, Fadwa; Bun, P; Verpillat, Frédéric; Suck, Sarah Y; Tessier, G; Atlan, Michael; Desbiolles, Pierre; Coppey-Moisan, Maite; Abboud, Marie; Gross, Michel

    2011-01-01

    In this paper, we explore the 3D structure of light scattering from dark-field illuminated live 3T3 cells in the presence of 40 nm gold nanomarkers. For this purpose, we use a high resolution holographic microscope combining the off-axis heterodyne geometry and the phase-shifting acquisition of the digital holograms. A comparative study of the 3D reconstructions of the scattered fields allows us to locate the gold markers which yield, contrarily to the cell structures, well defined bright scattering patterns that are not angularly titled and clearly located along the optical axis (z). This characterization is an unambiguous signature of the presence of gold biological nanomarkers, and validates the capability of digital holographic microscopy to discriminate them from background signals in live ce

  8. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

    Science.gov (United States)

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-12-01

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

  9. Rearrangements between differentiating hair cells coordinate planar polarity and the establishment of mirror symmetry in lateral-line neuromasts

    Directory of Open Access Journals (Sweden)

    Ivana Mirkovic

    2012-04-01

    In addition to their ubiquitous apical-basal polarity, many epithelia are also polarized along an orthogonal axis, a phenomenon termed planar cell polarity (PCP. In the mammalian inner ear and the zebrafish lateral line, PCP is revealed through the orientation of mechanosensitive hair cells relative to each other and to the body axes. In each neuromast, the receptor organ of the lateral line, hair bundles are arranged in a mirror-symmetrical fashion. Here we show that the establishment of mirror symmetry is preceded by rotational rearrangements between hair-cell pairs, a behavior consistently associated with the division of hair-cell precursors. Time-lapse imaging of trilobite mutants, which lack the core PCP constituent Vang-like protein 2 (Vangl2, shows that their misoriented hair cells correlate with misaligned divisions of hair-cell precursors and an inability to complete rearrangements accurately. Vangl2 is asymmetrically localized in the cells of the neuromast, a configuration required for accurate completion of rearrangements. Manipulation of Vangl2 expression or of Notch signaling results in a uniform hair-cell polarity, indicating that rearrangements refine neuromast polarity with respect to the body axes.

  10. Role of nanotopography in the development of tissue engineered 3D organs and tissues using mesenchymal stem cells

    OpenAIRE

    Salmasi, Shima; Kalaskar, Deepak M.; Yoon, Wai-Weng; Blunn, Gordon W.; Seifalian, Alexander M

    2015-01-01

    Recent regenerative medicine and tissue engineering strategies (using cells, scaffolds, medical devices and gene therapy) have led to fascinating progress of translation of basic research towards clinical applications. In the past decade, great deal of research has focused on developing various three dimensional (3D) organs, such as bone, skin, liver, kidney and ear, using such strategies in order to replace or regenerate damaged organs for the purpose of maintaining or restoring organs’ func...

  11. 3D Lifetime Tomography Reveals How CdCl 2 Improves Recombination Throughout CdTe Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Barnard, Edward S. [Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; PLANT PV, Inc, Alameda CA 94501 USA; Ursprung, Benedikt [PLANT PV, Inc, Alameda CA 94501 USA; Colegrove, Eric [National Renewable Energy Laboratory, Golden CO 80401 USA; Moutinho, Helio R. [National Renewable Energy Laboratory, Golden CO 80401 USA; Borys, Nicholas J. [Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; Hardin, Brian E. [PLANT PV, Inc, Alameda CA 94501 USA; Peters, Craig H. [PLANT PV, Inc, Alameda CA 94501 USA; Metzger, Wyatt K. [National Renewable Energy Laboratory, Golden CO 80401 USA; Schuck, P. James [Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA

    2016-11-15

    Using two-photon tomography, carrier lifetimes are mapped in polycrystalline CdTe photovoltaic devices. These 3D maps probe subsurface carrier dynamics that are inaccessible with traditional optical techniques. They reveal that CdCl2 treatment of CdTe solar cells suppresses nonradiative recombination and enhances carrier lifetimes throughout the film with substantial improvements particularly near subsurface grain boundaries and the critical buried p-n junction.

  12. Tuning the Mechanical Properties of Poly(Ethylene Glycol) Microgel-Based Scaffolds to Increase 3D Schwann Cell Proliferation.

    Science.gov (United States)

    Zhou, Wenda; Stukel, Jessica M; Cebull, Hannah L; Willits, Rebecca Kuntz

    2016-04-01

    2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel-based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol-ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈ 5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells.

    Science.gov (United States)

    Villinger, Clarissa; Gregorius, Heiko; Kranz, Christine; Höhn, Katharina; Münzberg, Christin; von Wichert, Götz; Mizaikoff, Boris; Wanner, Gerhard; Walther, Paul

    2012-10-01

    Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  18. Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

    Science.gov (United States)

    Shi, Pujiang; Laude, Augustinus; Yeong, Wai Yee

    2017-04-01

    In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017.

  19. Electricity Generation with the Novel 3D Electrode from Swim Wastewater in a Dual-chamber Microbial Fuel Cell

    Directory of Open Access Journals (Sweden)

    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%.

  20. Super-resolved 3-D imaging of live cells organelles from bright-field photon transmission micrographs

    CERN Document Server

    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...

  1. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells.

    Science.gov (United States)

    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.

  2. Mean deformation metrics for quantifying 3D cell-matrix interactions without requiring information about matrix material properties.

    Science.gov (United States)

    Stout, David A; Bar-Kochba, Eyal; Estrada, Jonathan B; Toyjanova, Jennet; Kesari, Haneesh; Reichner, Jonathan S; Franck, Christian

    2016-03-15

    Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamide- and polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress-strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix's displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels.

  3. 3D printed sample holder for in-operando EPR spectroscopy on high temperature polymer electrolyte fuel cells

    Science.gov (United States)

    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. A subset of prostatic basal cell carcinomas harbor the MYB rearrangement of adenoid cystic carcinoma.

    Science.gov (United States)

    Bishop, Justin A; Yonescu, Raluca; Epstein, Jonathan I; Westra, William H

    2015-08-01

    Adenoid cystic carcinoma (ACC) is a basaloid tumor consisting of myoepithelial and ductal cells typically arranged in a cribriform pattern. Adenoid cystic carcinoma is generally regarded as a form of salivary gland carcinoma, but it can arise from sites unassociated with salivary tissue. A rare form of prostate carcinoma exhibits ACC-like features; it is no longer regarded as a true ACC but rather as prostatic basal cell carcinoma (PBCC) and within the spectrum of basaloid prostatic proliferations. True ACCs often harbor MYB translocations resulting in the MYB-NFIB fusion protein. MYB analysis could clarify the true nature of prostatic carcinomas that exhibit ACC features and thus help refine the classification of prostatic basaloid proliferations. Twelve PBCCs were identified from the pathology consultation files of Johns Hopkins Hospital. The histopathologic features were reviewed, and break-apart fluorescence in situ hybridization for MYB was performed. All 12 cases exhibited prominent basaloid histology. Four were purely solid, 7 exhibited a cribriform pattern reminiscent of salivary ACC, and 1 had a mixed pattern. The MYB rearrangement was detected in 2 (29%) of 7 ACC-like carcinomas but in none (0%) of the 5 PBCCs with a prominent solid pattern. True ACCs can arise in the prostate as is evidenced by the presence of the characteristic MYB rearrangement. When dealing with malignant basaloid proliferations in the prostate, recommendations to consolidate ACCs with other tumor types may need to be reassessed, particularly in light of the rapidly advancing field of biologic therapy where the identification of tumor-specific genetic alterations presents novel therapeutic targets.

  5. The applications of 3D-hexagonal mesoporous SiO2 in proton exchange membranes of fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, B.; Tang, H.; Pan, M.; Chen, L.; Liang, C. [Wuhan Univ. of Technology, Wuhan (China). State Key Laboratory of Advanced Technology for Materials Synthesis and Processing

    2010-07-01

    The advantages of 3D hexagonal ordered mesoporous silica include a homogeneous ordered pore, a large surface area, narrow pore size distribution and excellent thermal stability. Hetropolyacids (HPAs) are high conductivity solid proton conductors that can be loaded onto the channel of mesoporous silica as a carrier for the proton exchange membrane of fuel cells. In this study, an ordered 3D hexagonal structure HPW/SiO{sub 2} nano composite materials was prepared using the sol-gel method with a nonionic surfactant (Brij-76) as the template. The HPW/SiO{sub 2} composite was characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM) and N2 adsorption-desorption. The study showed that the synthesis method can effectively make the HPW disperse uniformly in mesoporous framework, ensuring that the synthesized material has ordered mesoscopic structure. Impedance analysis revealed that the HPW/silica nano-materials have good proton conductivity without humidification at room temperature.

  6. Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

    Directory of Open Access Journals (Sweden)

    Alvaro Muñoz-López

    2016-10-01

    Full Text Available Induced pluripotent stem cells (iPSCs are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

  7. T-cell receptor gene rearrangement analysis: cutaneous T cell lymphoma, peripheral T cell lymphoma, and premalignant and benign cutaneous lymphoproliferative disorders.

    Science.gov (United States)

    Zelickson, B D; Peters, M S; Muller, S A; Thibodeau, S N; Lust, J A; Quam, L M; Pittelkow, M R

    1991-11-01

    T-cell receptor gene rearrangement analysis is a useful technique to detect clonality and determine lineage of lymphoid neoplasms. We examined 103 patients with mycosis fungoides, Sézary syndrome, peripheral T cell lymphoma, potentially malignant lymphoproliferative disorders including pre-Sézary syndrome, large plaque parapsoriasis, lymphomatoid papulosis and follicular mucinosis, and various benign inflammatory infiltrates. A clonal rearrangement was detected in skin samples in 20 of 24 patients with mycosis fungoides and in peripheral blood samples in 19 of 21 patients with Sézary syndrome. A clonal population was also detected in seven of eight cases classified as peripheral T cell lymphoma. The potentially malignant dermatoses tended to have clonal rearrangement, with the exception of large plaque parapsoriasis, and further follow-up is needed to correlate clonality with the disease course. These studies demonstrate the value of molecular genetics as an adjunct to morphology in the examination of patients with cutaneous lymphoproliferative disease.

  8. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model.

    Directory of Open Access Journals (Sweden)

    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.

  9. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

    Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan

    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 limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic...... 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....

  11. 3D collagen scaffolds coated with multiwalled carbon nanotubes: initial cell attachment to internal surface.

    Science.gov (United States)

    Hirata, Eri; Uo, Motohiro; Nodasaka, Yoshinobu; Takita, Hiroko; Ushijima, Natsumi; Akasaka, Tsukasa; Watari, Fumio; Yokoyama, Atsuro

    2010-05-01

    The cell adhesion in a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) was investigated. Immediately after seeding, the cells adhered to the inner surface of the MWCNT-coated sponge and a significantly larger number of cells were observed there than for a pure collagen sponge used as control. On the MWCNT-coated sponge, the cells appeared favorable adhesion and spread in the early stages in the center part of the sponge which cells rarely attached without MWCNT-coating. It was suggested that the physical structure of MWCNTs was effective for initial adhesion of cells from the result of serum-free culture. MWCNT-coating makes the material a suitable three-dimensional scaffold for cell culturing, as opposed to other scaffold systems where such an effect is not seen.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    Science.gov (United States)

    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.

  14. New process for fuel cell fabrication. 3D screen printing of metal bipolar plates; Neues Verfahren zur Brennstoffzellenfertigung. 3D-Siebdruck von metallischen Bipolarplatten

    Energy Technology Data Exchange (ETDEWEB)

    Studnitzky, Thomas [Fraunhofer-Institut fuer Fertigungstechnik und Angewandte Materialforschung (IFAM), Dresden (Germany); Helm, Peter; Heinzel, Angelika [Zentrum fuer BrennstoffzellenTechnik GmbH (ZBT), Duisburg (Germany)

    2011-01-15

    Minimization of space requirements, weight, and production cost is one of the key preconditions for successful launching of the polymer electrolyte membrane fuel cell (PEM). In the stacks constructed from single PEM cells, the bipolar plate is a central component. It determines the weight and volume of the stack and accounts for more than 30 percent of the overall cost, depending on the fabrication process. It is therefore important for producers of fuel cells to develop a process that combines free design, high functionality and low cost in serial production.

  15. Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations

    Energy Technology Data Exchange (ETDEWEB)

    Douglass, Michael; Bezak, Eva; Penfold, Scott [School of Chemistry and Physics, University of Adelaide, North Terrace, Adelaide 5005, South Australia (Australia) and Department of Medical Physics, Royal Adelaide Hospital, North Terrace, Adelaide 5000, South Australia (Australia)

    2012-06-15

    Purpose: The objective of the current work was to develop an algorithm for growing a macroscopic tumor volume from individual randomized quasi-realistic cells. The major physical and chemical components of the cell need to be modeled. It is intended to import the tumor volume into GEANT4 (and potentially other Monte Carlo packages) to simulate ionization events within the cell regions. Methods: A MATLAB Copyright-Sign code was developed to produce a tumor coordinate system consisting of individual ellipsoidal cells randomized in their spatial coordinates, sizes, and rotations. An eigenvalue method using a mathematical equation to represent individual cells was used to detect overlapping cells. GEANT4 code was then developed to import the coordinate system into GEANT4 and populate it with individual cells of varying sizes and composed of the membrane, cytoplasm, reticulum, nucleus, and nucleolus. Each region is composed of chemically realistic materials. Results: The in-house developed MATLAB Copyright-Sign code was able to grow semi-realistic cell distributions ({approx}2 Multiplication-Sign 10{sup 8} cells in 1 cm{sup 3}) in under 36 h. The cell distribution can be used in any number of Monte Carlo particle tracking toolkits including GEANT4, which has been demonstrated in this work. Conclusions: Using the cell distribution and GEANT4, the authors were able to simulate ionization events in the individual cell components resulting from 80 keV gamma radiation (the code is applicable to other particles and a wide range of energies). This virtual microdosimetry tool will allow for a more complete picture of cell damage to be developed.

  16. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

    OpenAIRE

    1993-01-01

    Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow...

  17. Numerical estimation of 3D mechanical forces exerted by cells on non-linear materials.

    Science.gov (United States)

    Palacio, J; Jorge-Peñas, A; Muñoz-Barrutia, A; Ortiz-de-Solorzano, C; de Juan-Pardo, E; García-Aznar, J M

    2013-01-04

    The exchange of physical forces in both cell-cell and cell-matrix interactions play a significant role in a variety of physiological and pathological processes, such as cell migration, cancer metastasis, inflammation and wound healing. Therefore, great interest exists in accurately quantifying the forces that cells exert on their substrate during migration. Traction Force Microscopy (TFM) is the most widely used method for measuring cell traction forces. Several mathematical techniques have been developed to estimate forces from TFM experiments. However, certain simplifications are commonly assumed, such as linear elasticity of the materials and/or free geometries, which in some cases may lead to inaccurate results. Here, cellular forces are numerically estimated by solving a minimization problem that combines multiple non-linear FEM solutions. Our simulations, free from constraints on the geometrical and the mechanical conditions, show that forces are predicted with higher accuracy than when using the standard approaches.

  18. Diagnosis of mantle cell lymphoma and detection of bcl-1 gene rearrangement

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Sook; Cho, Kyung Ja; Lee, Sun Joo [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    We reclassified a large series of non-Hogkin`s lymphoma diagnosed at Korea Cancer Center Hospital from 1991 to 1995, according to REAL classification, and compared the efficacy of immunohistochemical study for cyclin D1 protein and PCR for bcl-1 gene rearrangement to diagnose mantle cell lymphoma (MCL). By REAL classification, 7 %, diffuse large B-cell lymphoma was the most common type (51.8%) and was followed by peripheral T-cell lymphoma-unspecified (10%) and angiocentric lymphoma (7.5%). The most reliable histologic finding was mitosis to make a differential diagnosis. Mitoses of MCL were 17/10 HPF in average and all the cases showed more than 10/10 HPF. Immunophenotypic study alone cannot lead to a differential diagnosis between MCL and SLL, and the overexpression of cyclin D1 was the most important for diagnosis of MCL . Both immunohistochemistry for cyclin D1 and PCR for bcl-1 were specific for MCL and immunohistochemistry was more sensitive than PCR. Statistical analysis showed a different survival rate between MCL and the other low-grade B-cell lymphomas (SLL + MALT + LPL) and a difference between MCL and SLL. Immunohistochemical detection of cyclin D1 has a practical usefulness in making routine diagnosis of MCL. The initial accurate diagnosis of MCL will help clinicians make a proper management. (author). 27 refs., 6 tabs., 4 figs.

  19. Inhibition of RORγT Skews TCRα Gene Rearrangement and Limits T Cell Repertoire Diversity

    Directory of Open Access Journals (Sweden)

    Yanxia Guo

    2016-12-01

    Full Text Available Recent studies have elucidated the molecular mechanism of RORγT transcriptional regulation of Th17 differentiation and function. RORγT was initially identified as a transcription factor required for thymopoiesis by maintaining survival of CD4+CD8+ (DP thymocytes. While RORγ antagonists are currently being developed to treat autoimmunity, it remains unclear how RORγT inhibition may impact thymocyte development. In this study, we show that in addition to regulating DP thymocytes survival, RORγT also controls genes that regulate thymocyte migration, proliferation, and T cell receptor (TCRα selection. Strikingly, pharmacological inhibition of RORγ skews TCRα gene rearrangement, limits T cell repertoire diversity, and inhibits development of autoimmune encephalomyelitis. Thus, targeting RORγT not only inhibits Th17 cell development and function but also fundamentally alters thymic-emigrant recognition of self and foreign antigens. The analysis of RORγ inhibitors has allowed us to gain a broader perspective of the diverse function of RORγT and its impact on T cell biology.

  20. CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis.

    Science.gov (United States)

    Collins, Carl; Maruthi, N M; Jahn, Courtney E

    2015-08-01

    A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants.

  1. Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.

    NARCIS (Netherlands)

    Droese, J.; Langerak, A.W.; Groenen, P.J.T.A.; Bruggemann, M.; Neumann, P.; Wolvers-Tettero, I.L.M.; Altena, M.C. van; Kneba, M.; Dongen, J.J.M. van

    2004-01-01

    The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB)

  2. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma

    NARCIS (Netherlands)

    Kramer, M.H.H.; Hermans, J; Wijburg, E; Philippo, K; Geelen, E; van Krieken, J.H.J.M.; de Jong, D; Maartense, E; Schuuring, E; Kluin, P M

    1998-01-01

    Diffuse large B-cell lymphoma (DLCL) is characterized by a marked degree of morphologic and clinical heterogeneity. We studied 156 patients with de novo DLCL for rearrangements of the BCL2, BCL6, and MYC oncogenes by Southern blot analysis and BCL2 protein expression. We related these data to the pr

  3. A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

    Science.gov (United States)

    Sabra, Georges; Vermette, Patrick

    2013-02-01

    The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered.

  4. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xionggao [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Department of Ophthalmology, Hainan Medical College, Haikou (China); Wei, Yantao; Ma, Haizhi [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Zhang, Shaochong, E-mail: zhshaochong@163.com [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  5. Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

    Science.gov (United States)

    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.

  6. A general approach for time-supersampling of 3D-PIV data by the vortex-in-cell method

    Science.gov (United States)

    Scarano, Fulvio; Schneiders, Jan; Dwight, Richard; Aerospace Engineering/Aerodynamics Team

    2013-11-01

    Advancements of tomographic PIV [1] have led into 3D time-resolved experiments to study the dynamical evolution of 3D turbulent flows [2]. The known bottleneck of Tomo-PIV is the high laser power required to illuminate large volumes in airflows, which becomes critical beyond 10 kHz. Time-super-sampling is an approach to reduce the sampling rate, proven for frozen turbulence where the advection model yields a significant increase of temporal resolution [3]. Instead, in separated flows, the advection principle yields unacceptable distortions. The use of Navier-Stokes numerical calculations with the vortex-in-cell (VIC) method is proposed herein. The assumption is made of inviscid incompressible flow [4]. The spatial-resolution of the data is exploited to increase the temporal resolution. The dynamical evolution of the vorticity and velocity field between subsequent snapshots in the 3D domain is numerically evaluated. The verification with fully time resolved data of a circular jet indicates a substantial increase of temporal resolution. Interestingly, data sampled below the Nyquist limit could be reconstructed faithfully, indicating the potential of VIC in alleviating requirements on PIV measurement rate. Work supported by the European Research Council grant 202887.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Levato, Riccardo; Visser, Jetze; Planell, Josep A; Engel, Elisabeth; Malda, Jos; Mateos-Timoneda, Miguel A

    2014-09-01

    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.

  9. Biofunctionalized 3-D Carbon Nano-Network Platform for Enhanced Fibroblast Cell Adhesion

    Science.gov (United States)

    Chowdhury, A. K. M. Rezaul Haque; Tavangar, Amirhossein; Tan, Bo; Venkatakrishnan, Krishnan

    2017-01-01

    Carbon nanomaterials have been investigated for various biomedical applications. In most cases, however, these nanomaterials must be functionalized biologically or chemically due to their biological inertness or possible cytotoxicity. Here, we report the development of a new carbon nanomaterial with a bioactive phase that significantly promotes cell adhesion. We synthesize the bioactive phase by introducing self-assembled nanotopography and altered nano-chemistry to graphite substrates using ultrafast laser. To the best of our knowledge, this is the first time that such a cytophilic bio-carbon is developed in a single step without requiring subsequent biological/chemical treatments. By controlling the nano-network concentration and chemistry, we develop platforms with different degrees of cell cytophilicity. We study quantitatively and qualitatively the cell response to nano-network platforms with NIH-3T3 fibroblasts. The findings from the in vitro study indicate that the platforms possess excellent biocompatibility and promote cell adhesion considerably. The study of the cell morphology shows a healthy attachment of cells with a well-spread shape, overextended actin filaments, and morphological symmetry, which is indicative of a high cellular interaction with the nano-network. The developed nanomaterial possesses great biocompatibility and considerably stimulates cell adhesion and subsequent cell proliferation, thus offering a promising path toward engineering various biomedical devices. PMID:28287138

  10. An Innovative Hybrid 3D Analytic-Numerical Approach for System Level Modelling of PEM Fuel Cells

    Directory of Open Access Journals (Sweden)

    Gregor Tavčar

    2013-10-01

    Full Text Available The PEM fuel cell model presented in this paper is based on modelling species transport and coupling electrochemical reactions to species transport in an innovative way. Species transport is modelled by obtaining a 2D analytic solution for species concentration distribution in the plane perpendicular to the gas-flow and coupling consecutive 2D solutions by means of a 1D numerical gas-flow model. The 2D solution is devised on a jigsaw puzzle of multiple coupled domains which enables the modelling of parallel straight channel fuel cells with realistic geometries. Electrochemical and other nonlinear phenomena are coupled to the species transport by a routine that uses derivative approximation with prediction-iteration. A hybrid 3D analytic-numerical fuel cell model of a laboratory test fuel cell is presented and evaluated against a professional 3D computational fluid dynamic (CFD simulation tool. This comparative evaluation shows very good agreement between results of the presented model and those of the CFD simulation. Furthermore, high accuracy results are achieved at computational times short enough to be suitable for system level simulations. This computational efficiency is owed to the semi-analytic nature of its species transport modelling and to the efficient computational coupling of electrochemical kinetics and species transport.

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

    Science.gov (United States)

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

    2016-02-28

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

  12. Neuronal cells' behavior on polypyrrole coated bacterial nanocellulose three-dimensional (3D) scaffolds.

    Science.gov (United States)

    Muller, D; Silva, J P; Rambo, C R; Barra, G M O; Dourado, F; Gama, F M

    2013-01-01

    In this work, polypyrrole (PPy) was in situ polymerized onto the surface of bacterial nanocellulose (BNC) produced by Gluconacetobacter xylinus, by chemical oxidation in aqueous medium using ammonium persulfate. Composites (BNC/PPy) were produced with varying concentrations of pyrrole (Py). The produced BNC/PPy membranes were used as a template for the seeding of PC12 rat neuronal cells. Cell suspensions were directly seeded onto the surfaces of the BNC/PPy membranes. The Py concentration affected the behavior of neuronal cells that adhered and grew significantly more on BNC/PPy comparatively to BNC. Scanning electron microscopy (SEM) micrographs revealed that PC12 cells adhered on the surface of the BNC and BNC/PPy membranes. Conductive PPy coatings on nanofibers acting as an active interface for tissue engineering may be used to regulate cell activity through electrical stimulations.

  13. 3D Bearing Capacity of Structured Cells Supported on Cohesive Soil: Simplified Analysis Method

    Directory of Open Access Journals (Sweden)

    Martínez-Galván Sergio Antonio

    2013-06-01

    Full Text Available In this paper a simplified analysis method to compute the bearing capacity of structured cell foundations subjected to vertical loading and supported in soft cohesive soil is proposed. A structured cell is comprised by a top concrete slab structurally connected to concrete external walls that enclose the natural soil. Contrary to a box foundation it does not include a bottom slab and hence, the soil within the walls becomes an important component of the structured cell. This simplified method considers the three-dimensional geometry of the cell, the undrained shear strength of cohesive soils and the existence of structural continuity between the top concrete slab and the surrounding walls, along the walls themselves and the walls structural joints. The method was developed from results of numerical-parametric analyses, from which it was found that structured cells fail according to a punching-type mechanism.

  14. Enrichment of diluted cell populations from large sample volumes using 3D carbon-electrode dielectrophoresis.

    Science.gov (United States)

    Islam, Monsur; Natu, Rucha; Larraga-Martinez, Maria Fernanda; Martinez-Duarte, Rodrigo

    2016-05-01

    Here, we report on an enrichment protocol using carbon electrode dielectrophoresis to isolate and purify a targeted cell population from sample volumes up to 4 ml. We aim at trapping, washing, and recovering an enriched cell fraction that will facilitate downstream analysis. We used an increasingly diluted sample of yeast, 10(6)-10(2) cells/ml, to demonstrate the isolation and enrichment of few cells at increasing flow rates. A maximum average enrichment of 154.2 ± 23.7 times was achieved when the sample flow rate was 10 μl/min and yeast cells were suspended in low electrically conductive media that maximizes dielectrophoresis trapping. A COMSOL Multiphysics model allowed for the comparison between experimental and simulation results. Discussion is conducted on the discrepancies between such results and how the model can be further improved.

  15. Fabrication and characterization of microsieve electrode array (µSEA) enabling cell positioning on 3D electrodes

    Science.gov (United States)

    Schurink, B.; Tiggelaar, R. M.; Gardeniers, J. G. E.; Luttge, R.

    2017-01-01

    Here the fabrication and characterization of a novel microelectrode array for electrophysiology applications is described, termed a micro sieve electrode array (µSEA). This silicon based µSEA device allows for hydrodynamic parallel positioning of single cells on 3D electrodes realized on the walls of inverted pyramidal shaped pores. To realize the µSEA, a previously realized silicon sieving structure is provided with a patterned boron doped poly-silicon, connecting the contact electrodes with the 3D sensing electrodes in the pores. A LPCVD silicon-rich silicon nitride layer was used as insulation. The selective opening of this insulation layer at the ends of the wiring lines allows to generate well-defined contact and sensing electrodes according to the layout used in commercial microelectrode array readers. The main challenge lays in the simultaneously selective etching of material at both the planar surface (contact electrode) as well as in the sieving structure containing the (3D) pores (sensing electrodes). For the generation of 3D electrodes in the pores a self-aligning technique was developed using the pore geometry to our advantage. This technique, based on sacrificial layer etching, allows for the fine tuning of the sensing electrode surface area and thus supports the positioning and coupling of single cells on the electrode surface in relation to the cell size. Furthermore, a self-aligning silicide is formed on the sensing electrodes to favour the electrical properties. Experiments were performed to demonstrate the working principle of the µSEA using different types of neuronal cells. Capture efficiency in the pores was  >70% with a 70% survival rate of the cell maintained for up to 14 DIV. The TiSi2-boron-doped-poly-silicon sensing electrodes of the µSEA were characterized, which indicated noise levels of  <15 µV and impedance values of 360 kΩ. These findings potentially allow for future electrophysiological measurements using the µSEA.

  16. High resolution 3D imaging of living cells with sub-optical wavelength phonons

    Science.gov (United States)

    Pérez-Cota, Fernando; Smith, Richard J.; Moradi, Emilia; Marques, Leonel; Webb, Kevin F.; Clark, Matt

    2016-12-01

    Label-free imaging of living cells below the optical diffraction limit poses great challenges for optical microscopy. Biologically relevant structural information remains below the Rayleigh limit and beyond the reach of conventional microscopes. Super-resolution techniques are typically based on the non-linear and stochastic response of fluorescent labels which can be toxic and interfere with cell function. In this paper we present, for the first time, imaging of live cells using sub-optical wavelength phonons. The axial imaging resolution of our system is determined by the acoustic wavelength (λa = λprobe/2n) and not on the NA of the optics allowing sub-optical wavelength acoustic sectioning of samples using the time of flight. The transverse resolution is currently limited to the optical spot size. The contrast mechanism is significantly determined by the mechanical properties of the cells and requires no additional contrast agent, stain or label to image the cell structure. The ability to breach the optical diffraction limit to image living cells acoustically promises to bring a new suite of imaging technologies to bear in answering exigent questions in cell biology and biomedicine.

  17. A novel diamond anvil cell for x-ray diffraction at cryogenic temperatures manufactured by 3D printing

    Science.gov (United States)

    Jin, H.; Woodall, C. H.; Wang, X.; Parsons, S.; Kamenev, K. V.

    2017-03-01

    A new miniature high-pressure diamond anvil cell was designed and constructed using 3D micro laser sintering technology. This is the first application of the use of rapid prototyping technology to construct high-pressure apparatus. The cell is specifically designed for use as an X-ray diffraction cell that can be used with commercially available diffractometers and open-flow cryogenic equipment to collect data at low temperature and high pressure. The cell is constructed from stainless steel 316L and is about 9 mm in diameter and 7 mm in height, giving it both small dimensions and low thermal mass, and it will fit into the cooling envelope of a standard CryostreamTM cooling system. The cell is clamped using a customized miniature buttress thread of diameter 7 mm and pitch of 0.5 mm enabled by 3D micro laser sintering technology; such dimensions are not attainable using conventional machining. The buttress thread was used as it has favourable uniaxial load properties allowing for higher pressure and better anvil alignment. The clamp can support the load of at least 1.5 kN according to finite element analysis (FEA) simulations. FEA simulations were also used to compare the performance of the standard thread and the buttress thread, and demonstrate that stress is distributed more uniformly in the latter. Rapid prototyping of the pressure cell by the laser sintering resulted in a substantially higher tensile yield strength of the 316L stainless steel (675 MPa compared to 220 MPa for the wrought type of the same material), which increased the upper pressure limit of the cell. The cell is capable of reaching pressures of up to 15 GPa with 600 μm diameter culets of diamond anvils. Sample temperature and pressure changes on cooling were assessed using X-ray diffraction on samples of NaCl and HMT-d12.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    through smaller pore sizes made significantly more turns than those through larger pores. The introduction of additional defined barriers in the microporous structure resulted in dendritic cells making more turns while still being able to follow the chemoattractant concentration gradient....

  19. A 3-D Model of Signaling and Transport Pathways in Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    2016-03-18

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

  1. Passivation of ZnO Nanowire Guests and 3D Inverse Opal Host Photoanodes for Dye-Sensitized Solar Cells

    KAUST Repository

    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.

  2. The influence of plasma technology coupled to chemical grafting on the cell growth compliance of 3D hydroxyapatite scaffolds.

    Science.gov (United States)

    Russo, Laura; Zanini, Stefano; Giannoni, Paolo; Landi, Elena; Villa, Anna; Sandri, Monica; Riccardi, Claudia; Quarto, Rodolfo; Doglia, Silvia M; Nicotra, Francesco; Cipolla, Laura

    2012-11-01

    The development of advanced materials with biomimetic features in order to elicit desired biological responses and to guarantee tissue biocompatibility is recently gaining attention for tissue engineering applications. Bioceramics, such as hydroxyapatite-based biomaterials are now used in a number of different applications throughout the body, covering all areas of the skeleton, due to their biological and chemical similarity to the inorganic phases of bones. When bioactive sintered hydroxyapatite (HA) is desired, biomolecular modification of these materials is needed. In the present work, we investigated the influence of plasma surface modification coupled to chemical grafting on the cell growth compliance of HA 3D scaffolds.

  3. Differentiation capacity and maintenance of differentiated phenotypes of human mesenchymal stromal cells cultured on two distinct types of 3D polymeric scaffolds.

    Science.gov (United States)

    Leferink, A M; Santos, D; Karperien, M; Truckenmüller, R K; van Blitterswijk, C A; Moroni, L

    2015-12-01

    Many studies have shown the influence of soluble factors and material properties on the differentiation capacity of mesenchymal stromal cells (MSCs) cultured as monolayers. These types of two-dimensional (2D) studies can be used as simplified models to understand cell processes related to stem cell sensing and mechano-transduction in a three-dimensional (3D) context. For several other mechanisms such as cell-cell signaling, cell proliferation and cell morphology, it is well-known that cells behave differently on a planar surface compared to cells in 3D environments. In classical tissue engineering approaches, a combination of cells, 3D scaffolds and soluble factors are considered as the key ingredients for the generation of mechanically stable 3D tissue constructs. However, when MSCs are used for tissue engineering strategies, little is known about the maintenance of their differentiation potential in 3D scaffolds after the removal of differentiation soluble factors. In this study, the differentiation potential of human MSCs (hMSCs) into the chondrogenic and osteogenic lineages on two distinct 3D scaffolds, additive manufactured electrospun scaffolds, was assessed and compared to conventional 2D culture. Human MSCs cultured in the presence of soluble factors in 3D showed to differentiate to the same extent as hMSCs cultured as 2D monolayers or as scaffold-free pellets, indicating that the two scaffolds do not play a consistent role in the differentiation process. In the case of phenotypic changes, the achieved differentiated phenotype was not maintained after the removal of soluble factors, suggesting that the plasticity of hMSCs is retained in 3D cell culture systems. This finding can have implications for future tissue engineering approaches in which the validation of hMSC differentiation on 3D scaffolds will not be sufficient to ensure the maintenance of the functionality of the cells in the absence of appropriate differentiation signals.

  4. Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

    Directory of Open Access Journals (Sweden)

    JFW Greiner

    2011-12-01

    Full Text Available Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

  5. Preparation, characterization, and silanization of 3D microporous PDMS structure with properly sized pores for endothelial cell culture.

    Science.gov (United States)

    Zargar, Reyhaneh; Nourmohammadi, Jhamak; Amoabediny, Ghassem

    2016-01-01

    Nowadays, application of porous polydimethylsiloxane (PDMS) structure in biomedical is becoming widespread, and many methods have been established to create such structure. Although the pores created through these methods are mostly developed on the outer surface of PDMS membrane, this study offers a simple and cost-efficient technique for creating three-dimensional (3D) microporous PDMS structure with appropriate pore size for endothelial cell culture. In this study, combination of gas foaming and particulate leaching methods, with NaHCO3 as effervescent salt and NaCl as progen are used to form a 3D PDMS sponge. The in situ chemical reaction between NaHCO3 and HCl resulted in the formation of small pores and channels. Moreover, soaking the samples in HCl solution temporarily improved the hydrophilicity of PDMS, which then facilitated the penetration of water for further leaching of NaCl. The surface chemical modification process was performed by (3-aminopropyl)triethoxysilane to culture endothelial cells on porous PDMS matrix. The results are an indication of positive response of endothelial cells to the fabricated PDMS sponge. Because of simplicity and practicality of this method for preparing PDMS sponge with appropriate pore size and biological properties, the fabricated matrix can perfectly be applied to future studies in blood-contacting devices.

  6. A new 3-D ray tracing method based on LTI using successive partitioning of cell interfaces and traveltime gradients

    Science.gov (United States)

    Zhang, Dong; Zhang, Ting-Ting; Zhang, Xiao-Lei; Yang, Yan; Hu, Ying; Qin, Qian-Qing

    2013-05-01

    We present a new method of three-dimensional (3-D) seismic ray tracing, based on an improvement to the linear traveltime interpolation (LTI) ray tracing algorithm. This new technique involves two separate steps. The first involves a forward calculation based on the LTI method and the dynamic successive partitioning scheme, which is applied to calculate traveltimes on cell boundaries and assumes a wavefront that expands from the source to all grid nodes in the computational domain. We locate several dynamic successive partition points on a cell's surface, the traveltimes of which can be calculated by linear interpolation between the vertices of the cell's boundary. The second is a backward step that uses Fermat's principle and the fact that the ray path is always perpendicular to the wavefront and follows the negative traveltime gradient. In this process, the first-arriving ray path can be traced from the receiver to the source along the negative traveltime gradient, which can be calculated by reconstructing the continuous traveltime field with cubic B-spline interpolation. This new 3-D ray tracing method is compared with the LTI method and the shortest path method (SPM) through a number of numerical experiments. These comparisons show obvious improvements to computed traveltimes and ray paths, both in precision and computational efficiency.

  7. Response of microscale cell/matrix constructs to successive force application in a 3D environment

    Science.gov (United States)

    Liu, Alan; Chen, Christopher; Reich, Daniel

    2014-03-01

    Mechanical dilation of arteries by pulsatile blood flow is directly opposed by coordinated contraction of a band of smooth muscle tissue that envelops the vessels. This mechanical adaptation of smooth muscle cells to external loading is a critical feature of normal blood vessel function. While most previous studies on biomechanical systems have focused on single cells or large excised tissue, we utilize a device to apply forces to engineered smooth muscle microtissues. This device consists of arrayed pairs of elastomeric micro-cantilevers capable of magnetic actuation. Tissues are formed through self-assembly following the introduction of cell-infused collagen gel to the array. With this system, we are able to dynamically stretch and relax these sub-millimeter sized tissues. The timing and magnitude of the force application can be precisely controlled and thus can be used to mimic a wide range of physiological behavior. In particular, we will discuss results that show that the interval between successive force applications mediates the both the subsequent mechanical and active dynamics of the cell/matrix composite system. Understanding this process will lead to better understanding of the interplay between cell and extracellular matrix responses to mechanical stimulus at a novel length scale.

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

    Directory of Open Access Journals (Sweden)

    Gerald Burgstaller

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

  9. Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines.

    Science.gov (United States)

    Cao, Yihua; Kimura, Shuichi; Itoi, Takayuki; Honda, Kohsuke; Ohtake, Hisao; Omasa, Takeshi

    2012-06-01

    Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.

  10. Simulation of 3D tumor cell growth using nonlinear finite element method.

    Science.gov (United States)

    Dong, Shoubing; Yan, Yannan; Tang, Liqun; Meng, Junping; Jiang, Yi

    2016-01-01

    We propose a novel parallel computing framework for a nonlinear finite element method (FEM)-based cell model and apply it to simulate avascular tumor growth. We derive computation formulas to simplify the simulation and design the basic algorithms. With the increment of the proliferation generations of tumor cells, the FEM elements may become larger and more distorted. Then, we describe a remesh and refinement processing of the distorted or over large finite elements and the parallel implementation based on Message Passing Interface to improve the accuracy and efficiency of the simulation. We demonstrate the feasibility and effectiveness of the FEM model and the parallelization methods in simulations of early tumor growth.

  11. Discrete Element Modeling Results of Proppant Rearrangement in the Cooke Conductivity Cell

    Energy Technology Data Exchange (ETDEWEB)

    Earl Mattson; Hai Huang; Michael Conway; Lisa O' Connell

    2014-02-01

    The study of propped fracture conductivity began in earnest with the development of the Cooke cell which later became part of the initial API standard. Subsequent developments included a patented multicell design to conduct 4 tests in a press at the same time. Other modifications have been used by various investigators. Recent studies by the Stim-Lab proppant consortium have indicated that the flow field across a Cooke proppant conductivity testing cell may not be uniform as initially believed which resulted is significantly different conductivity results. Post test analysis of low temperature metal alloy injections at the termination of proppant testing prior to the release of the applied stress suggest that higher flow is to be expected along the sides and top of the proppant pack than compared to the middle of the pack. To evaluate these experimental findings, a physics-based two-dimensional (2-D) discrete element model (DEM) was developed and applied to simulate proppant rearrangement during stress loading in the Cooke conductivity cell and the resulting porosity field. Analysis of these simulations are critical to understanding the impact of modification to the testing cell as well as understanding key proppant conductivity issues such as how these effects are manifested in proppant concentration testing results. The 2-D DEM model was constructed to represent a realistic cross section of the Cooke cell with a distribution of four material properties, three that represented the Cooke cell (steel, sandstone,square rings), and one representing the proppant. In principle, Cooke cell materials can be approximated as assemblies of independent discrete elements (particles) of various sizes and material properties that interact via cohesive interactions, repulsive forces, and frictional forces. The macroscopic behavior can then be modeled as the collective behavior of many interacting discrete elements. This DEM model is particularly suitable for modeling proppant

  12. 3D Cell Culture in a Self-Assembled Nanofiber Environment

    Science.gov (United States)

    Gubbe, John D.; Brekke, John H.

    2016-01-01

    The development and utilization of three-dimensional cell culture platforms has been gaining more traction. Three-dimensional culture platforms are capable of mimicking in vivo microenvironments, which provide greater physiological relevance in comparison to conventional two-dimensional cultures. The majority of three-dimensional culture platforms are challenged by the lack of cell attachment, long polymerization times, and inclusion of undefined xenobiotics, and cytotoxic cross-linkers. In this study, we review the use of a highly defined material composed of naturally occurring compounds, hyaluronic acid and chitosan, known as Cell-Mate3DTM. Moreover, we provide an original measurement of Young’s modulus using a uniaxial unconfined compression method to elucidate the difference in microenvironment rigidity for acellular and cellular conditions. When hydrated into a tissue-like hybrid hydrocolloid/hydrogel, Cell-Mate3DTM is a highly versatile three-dimensional culture platform that enables downstream applications such as flow cytometry, immunostaining, histological staining, and functional studies to be applied with relative ease. PMID:27632425

  13. Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)–chitosan scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jing; Zhou, Aimei; Deng, Aipeng [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Yang [Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Gao, Lihu; Zhong, Zhaocai [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Shulin, E-mail: yshulin@njust.edu.cn [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2015-04-01

    Pore architecture of 3D scaffolds used in tissue engineering plays a critical role in the maintenance of cell survival, proliferation and further promotion of tissue regeneration. We investigated the pore size and structure, porosity, swelling as well as cell viability of a series of recombinant human collagen-peptide–chitosan (RHCC) scaffolds fabricated by lyophilization. In this paper, freezing regime containing a final temperature of freezing (T{sub f}) and cooling rates was applied to obtain scaffolds with pore size ranging from 100 μm to 120 μm. Other protocols of RHC/chitosan suspension concentration and ratio modification were studied to produce more homogenous and appropriate structural scaffolds. The mean pore size decreased along with the decline of T{sub f} at a slow cooling rate of 0.7 °C/min; a more rapid cooling rate under 5 °C/min resulted to a smaller pore size and more homogenous microstructure. High concentration could reduce pore size and lead to thick well of scaffold, while improved the ratio of RHC, lamellar and fiber structure coexisted with cellular pores. Human umbilical vein endothelial cells (HUVECs) were seeded on these manufactured scaffolds, the cell viability represented a negative correlation to the pore size. This study provides an alternative method to fabricate 3D RHC–chitosan scaffolds with appropriate pores for potential tissue engineering. - Highlights: • Fabrication of recombinant human collagen-chitosan scaffolds by freezing drying • Influence of freeze drying protocols on lyophilized scaffolds • Pore size, microstructure, porosity, swelling and cell viability were compared. • The optimized porous scaffold is suitable for cell (HUVEC) seeding.

  14. Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca(2+) signaling in CD8(+) T cells.

    Science.gov (United States)

    Averbeck, Marco; Braun, Thorsten; Pfeifer, Gunther; Sleeman, Jonathan; Dudda, Jan; Martin, Stefan F; Kremer, Bernhard; Aktories, Klaus; Simon, Jan C; Termeer, Christian

    2004-10-01

    The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca(2+) influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8(+) T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca(2+) influx in T cells to long-lasting interactions and sustained Ca(2+) influx of 30 min and more. Follow-up of DC/T cell interactions after 2 h using confocal microscopy revealed that long-lasting Ca(2+) responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-K(b) at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca(2+) stimulators 7 h after initiation of maturation. Instead, the enhanced capacity of 7 h-matured DC to induce sustained Ca(2+) responses in CD8(+) T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8(+) T cell stimulation.

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

    Science.gov (United States)

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

    2014-05-01

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

  16. Effect of 3D stall-cells on the pressure distribution of a laminar NACA64-418 wing

    Science.gov (United States)

    Ragni, Daniele; Ferreira, Carlos

    2016-08-01

    A 3D stall-cell flow-field has been studied in a 4.8 aspect-ratio wing obtained by linear extrusion of a laminar NACA64-418 airfoil profile. The span-wise change in the velocity and pressure distribution along the wing has been quantified with respect to the development of cellular structures from 8° to 20° angle of attack. Oil-flow visualizations help localizing the regular cellular pattern in function of the angle of attack. Multi-plane stereoscopic PIV measurements obtained by traversing the entire setup along the wing span show that the flow separation is not span-wise uniform. The combination of different stereoscopic fields into a 3D volume of velocity data allows studying the global effect of the stall-cell pattern on the wing flow. Integration of the experimentally computed pressure gradient from the Navier-Stokes equation is employed to compute the span-wise distribution of the mean surface pressure. Comparison of the results with the ones obtained from pressure taps installed in the wing evidences a span-wise periodic loading on the wing. The periodic loading has maxima confined in the stream-wise direction between the location of the highest airfoil curvature and the one of the airfoil flow separation. Estimation of the periodic loading is found within 2-6 % of the sectional wing lift.

  17. Automated detection of retinal cell nuclei in 3D micro-CT images of zebrafish using support vector machine classification

    Science.gov (United States)

    Ding, Yifu; Tavolara, Thomas; Cheng, Keith

    2016-03-01

    Our group is developing a method to examine biological specimens in cellular detail using synchrotron microCT. The method can acquire 3D images of tissue at micrometer-scale resolutions, allowing for individual cell types to be visualized in the context of the entire specimen. For model organism research, this tool will enable the rapid characterization of tissue architecture and cellular morphology from every organ system. This characterization is critical for proposed and ongoing "phenome" projects that aim to phenotype whole-organism mutants and diseased tissues from different organisms including humans. With the envisioned collection of hundreds to thousands of images for a phenome project, it is important to develop quantitative image analysis tools for the automated scoring of organism phenotypes across organ systems. Here we present a first step towards that goal, demonstrating the use of support vector machines (SVM) in detecting retinal cell nuclei in 3D images of wild-type zebrafish. In addition, we apply the SVM classifier on a mutant zebrafish to examine whether SVMs can be used to capture phenotypic differences in these images. The longterm goal of this work is to allow cellular and tissue morphology to be characterized quantitatively for many organ systems, at the level of the whole-organism.

  18. Changes in morphology of actin filaments and expression of alkaline phosphatase at 3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds.

    Science.gov (United States)

    Goncharenko, A V; Malyuchenko, N V; Moisenovich, A M; Kotlyarova, M S; Arkhipova, A Yu; Kon'kov, A S; Agapov, I I; Molochkov, A V; Moisenovich, M M; Kirpichnikov, M P

    2016-09-01

    3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.

  19. Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Engl

    2013-10-01

    Full Text Available Christopher G England,1 Thomas Priest,2 Guandong Zhang,2 Xinghua Sun,2 Dhruvinkumar N Patel,2 Lacey R McNally,3,4 Victor van Berkel,4,5 André M Gobin,2 Hermann B Frieboes1,2,41Department of Pharmacology and Toxicology, 2Department of Bioengineering, 3Department of Medicine, 4James Graham Brown Cancer Center, 5Department of Surgery, University of Louisville, KY, USAAbstract: Nano-scale particles sized 10–400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol (PEG-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.Keywords: cancer nanotherapy, tumor hypoxia, nanovector transport

  20. CONTROLLING THE 3D NANOSCALE ORGANIZATION OF BULK HETEROJUNCTION POLYMER SOLAR CELLS

    Institute of Scientific and Technical Information of China (English)

    Svetlana S. Van Bavel; Erwan Sourty; Gijsbertus de With; Joachim Loos

    2009-01-01

    In this study,the three dimensional nanoscale organization in the photoactive layers of poly(3-hexylthiophene)(P3HT) and a methanofullerene derivative (PCBM) is revealed by transmission electron tomography.After annealing treatment,either at elevated temperature or during slow solvent evaporation,nanoscale interpenetrating networks are formed with high crystalline order and favorable concentration gradients of both components through the thickness of the photoactive layer.Such a tailored morphology accounts for the considerable increase of the power conversion efficiency in corresponding solar cell devices.

  1. Cell therapy, 3D culture systems and tissue engineering for cardiac regeneration.

    Science.gov (United States)

    Emmert, Maximilian Y; Hitchcock, Robert W; Hoerstrup, Simon P

    2014-04-01

    Ischemic Heart Disease (IHD) still represents the "Number One Killer" worldwide accounting for the death of numerous patients. However the capacity for self-regeneration of the adult heart is very limited and the loss of cardiomyocytes in the infarcted heart leads to continuous adverse cardiac-remodeling which often leads to heart-failure (HF). The concept of regenerative medicine comprising cell-based therapies, bio-engineering technologies and hybrid solutions has been proposed as a promising next-generation approach to address IHD and HF. Numerous strategies are under investigation evaluating the potential of regenerative medicine on the failing myocardium including classical cell-therapy concepts, three-dimensional culture techniques and tissue-engineering approaches. While most of these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or too rapid leaving many key questions unanswered. This review summarizes the current state-of-the-art, important challenges and future research directions as to regenerative approaches addressing IHD and resulting HF.

  2. Numerical 3D-simulation of micromorph silicon thin film solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Geissendoerfer, Stefan; Lacombe, Juergen; Maydell, Karsten von; Agert, Carsten [EWE-Forschungszentrum fuer Energietechnologie e.V. NEXT ENERGY, Carl-von-Ossietzky-Str. 15, 26129 Oldenburg (Germany)

    2011-07-01

    In this contribution 3-dimensional simulations of micromorph silicon thin film solar cells, which have a tandem structure consisting of amorphous and microcrystalline subcells, are presented. The variety of different active layers leads to a very complex structure. Additionally, randomly textured surfaces and interfaces have to be taken into account. Our goal is to create physical models to describe the coupled optical and electrical behaviour of the whole structure in three dimensions to determine the theoretical limits and dominant material parameters. To simulate solar cells with rough interfaces, the surfaces topography was measured via atomic force microscopy (AFM) and transferred to the commercial software Sentaurus TCAD from the company Synopsys. The virtual structure includes layer thicknesses and optoelectronic parameters. Results of the space resolved optical generation rates by using the optical solver ''Raytracer'' are presented. The space resolved optical generation rate inside the semiconductor layers depends on the structure of the TCO interface. Therefore, regions with higher charge carrier densities can be observed which has an influence on the current transport through the stack. These investigations and the influence to the IV characteristic are presented.

  3. Cell and protein compatible 3D bioprinting of mechanically strong constructs for bone repair.

    Science.gov (United States)

    Sawkins, M J; Mistry, P; Brown, B N; Shakesheff, K M; Bonassar, L J; Yang, J

    2015-07-02

    Rapid prototyping of bone tissue engineering constructs often utilizes elevated temperatures, organic solvents and/or UV light for materials processing. These harsh conditions may prevent the incorporation of cells and therapeutic proteins in the fabrication processes. Here we developed a method for using bioprinting to produce constructs from a thermoresponsive microparticulate material based on poly(lactic-co-glycolic acid) at ambient conditions. These constructs could be engineered with yield stresses of up to 1.22 MPa and Young's moduli of up to 57.3 MPa which are within the range of properties of human cancellous bone. Further study showed that protein-releasing microspheres could be incorporated into the bioprinted constructs. The release of the model protein lysozyme from bioprinted constructs was sustainted for a period of 15 days and a high degree of protein activity could be measured up to day 9. This work suggests that bioprinting is a viable route to the production of mechanically strong constructs for bone repair under mild conditions which allow the inclusion of viable cells and active proteins.

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

    Science.gov (United States)

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

    2012-04-01

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

  5. Comparative proteomic analyses demonstrate enhanced Interferon and STAT-1 activation in reovirus T3D-infected HeLa cells

    Directory of Open Access Journals (Sweden)

    Peyman eEzzati

    2015-04-01

    Full Text Available As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC analyses of reovirus, strain type 3 Dearing (T3D-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D. Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26 and 44 were significantly down-regulated by T1L, T3D and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3 and Mx1. This study expands our understanding of reovirus-induced host responses.

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

    Science.gov (United States)

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

    2014-01-01

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

  7. Novel method to dynamically load cells in 3D-hydrogels culture for blast injury studies

    Science.gov (United States)

    Sory, David R.; Areias, Anabela C.; Overby, Darryl R.; Proud, William G.

    2017-01-01

    For at least a century explosive devices have been one of the most important causes of injuries in military conflicts as well as in terrorist attacks. Although significant experimental and modelling efforts have been focussed on blast injuries at the organ or tissue level, few studies have investigated the mechanisms of blast injuries at the cellular level. This paper introduces an in vitro method compatible with living cells to examine the effects of high stress and short-duration pulses relevant to blast loadings and blunt trauma. The experimental phase involves high strain-rate axial compression of cylindrical specimens within an hermetically sealed chamber made of biocompatible polymer. Numerical simulations were performed in order to verify the experimental loading conditions and to characterize the loading path within the sample. A proof of concept is presented so as to establish a new window to address fundamental questions regarding blast injury at the cellular level.

  8. Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures.

    Science.gov (United States)

    Ravi, Maddaly; Kaviya, S R; Paramesh, V

    2016-05-01

    Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.

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

    Science.gov (United States)

    Hosseinkhani, Hossein; Hong, Po-Da; Yu, Dah-Shyong; Chen, Yi-Ru; Ickowicz, Diana; Farber, Ira-Yudovin; Domb, Abraham J

    2012-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Elena Gaberman

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

  11. Enhanced hydrogen production in microbial electrolysis cell with 3D self-assembly nickel foam-graphene cathode.

    Science.gov (United States)

    Cai, Weiwei; Liu, Wenzong; Han, Jinglong; Wang, Aijie

    2016-06-15

    In comparison to precious metal catalyst especially Platinum (Pt), nickel foam (NF) owned cheap cost and unique three-dimensional (3D) structure, however, it was scarcely applied as cathode material in microbial electrolysis cell (MEC) as the intrinsic laggard electrochemical activity for hydrogen recovery. In this study, a self-assembly 3D nickel foam-graphene (NF-G) cathode was fabricated by facile hydrothermal approach for hydrogen evolution in MECs. Electrochemical analysis (linear scan voltammetry and electrochemical impedance spectroscopy) revealed the improved electrochemical activity and effective mass diffusion after coating with graphene. NF-G as cathode in MEC showed a significant enhancement in hydrogen production rate compared with nickel foam at a variety of biases. Noticeably, NF-G showed a comparable averaged hydrogen production rate (1.31 ± 0.07 mL H2 mL(-1) reactor d(-1)) to Platinum/carbon (Pt/C) (1.32 ± 0.07 mL H2 mL(-1) reactor d(-1)) at 0.8 V. Profitable energy recovery could be achieved by NF-G cathode at higher applied voltage, which performed the best hydrogen yield of 3.27 ± 0.16 mol H2 mol(-1) acetate at 0.8 V and highest energy efficiency of 185.92 ± 6.48% at 0.6 V.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

  14. On the Quality of Velocity Interpolation Schemes for Marker-In-Cell Methods on 3-D Staggered Grids

    Science.gov (United States)

    Kaus, B.; Pusok, A. E.; Popov, A.

    2015-12-01

    The marker-in-cell method is generally considered to be a flexible and robust method to model advection of heterogenous non-diffusive properties (i.e. rock type or composition) in geodynamic problems or incompressible Stokes problems. In this method, Lagrangian points carrying compositional information are advected with the ambient velocity field on an immobile, Eulerian grid. However, velocity interpolation from grid points to marker locations is often performed without preserving the zero divergence of the velocity field at the interpolated locations (i.e. non-conservative). Such interpolation schemes can induce non-physical clustering of markers when strong velocity gradients are present (Jenny et al., 2001) and this may, eventually, result in empty grid cells, a serious numerical violation of the marker-in-cell method. Solutions to this problem include: using larger mesh resolutions and/or marker densities, or repeatedly controlling the marker distribution (i.e. inject/delete), but which does not have an established physical background. To remedy this at low computational costs, Jenny et al. (2001) and Meyer and Jenny (2004) proposed a simple, conservative velocity interpolation (CVI) scheme for 2-D staggered grid, while Wang et al. (2015) extended the formulation to 3-D finite element methods. Here, we follow up with these studies and report on the quality of velocity interpolation methods for 2-D and 3-D staggered grids. We adapt the formulations from both Jenny et al. (2001) and Wang et al. (2015) for use on 3-D staggered grids, where the velocity components have different node locations as compared to finite element, where they share the same node location. We test the different interpolation schemes (CVI and non-CVI) in combination with different advection schemes (Euler, RK2 and RK4) and with/out marker control on Stokes problems with strong velocity gradients, which are discretized using a finite difference method. We show that a conservative formulation

  15. CAD/CAM-designed 3D-printed electroanalytical cell for the evaluation of nanostructured gas-diffusion electrodes

    Science.gov (United States)

    Chervin, Christopher N.; Parker, Joseph F.; Nelson, Eric S.; Rolison, Debra R.; Long, Jeffrey W.

    2016-04-01

    The ability to effectively screen and validate gas-diffusion electrodes is critical to the development of next-generation metal-air batteries and regenerative fuel cells. The limiting electrode in a classic two-terminal device such as a battery or fuel cell is difficult to discern without an internal reference electrode, but the flooded electrolyte characteristic of three-electrode electroanalytical cells negates the prime function of an air electrode—a void volume freely accessible to gases. The nanostructured catalysts that drive the energy-conversion reactions (e.g., oxygen reduction and evolution in the air electrode of metal-air batteries) are best evaluated in the electrode structure as-used in the practical device. We have designed, 3D-printed, and characterized an air-breathing, thermodynamically referenced electroanalytical cell that allows us to mimic the Janus arrangement of the gas-diffusion electrode in a metal-air cell: one face freely exposed to gases, the other wetted by electrolyte.

  16. Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel.

    Science.gov (United States)

    Wang, Zheng; Zhang, Yeshun; Zhang, Jinxiang; Huang, Lei; Liu, Jia; Li, Yongkui; Zhang, Guozheng; Kundu, Subhas C; Wang, Lin

    2014-11-20

    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 approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine.

  17. Correlating intravital multi-photon microscopy to 3D electron microscopy of invading tumor cells using anatomical reference points.

    Directory of Open Access Journals (Sweden)

    Matthia A Karreman

    Full Text Available Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.

  18. Rearrangement and expression of beta-T-cell receptor and immunoglobulin genes in established Ph1 chronic myelogenous leukemia cell lines.

    Science.gov (United States)

    Berenson, J; Koeffler, H P

    1989-01-01

    We have determined the arrangement and expression of immunoglobulin (Ig) and beta-T-cell receptor (TCR) genes in six established Philadelphia chromosome-positive (Ph1) chronic myelogenous leukemia (CML) cell lines, and correlated these results with their phenotypic characteristics. Three cell lines with nonlymphoid characteristics, EM2, EM3, and K562, did not demonstrate rearrangement or expression of Ig or beta-TCR genes. A new cell line, MB, with a mature B-cell phenotype recently established in our laboratory, contained light and heavy chain immunoglobulin gene rearrangements and expressed mature Ig RNA. In a cell line with an early lymphoid phenotype, BV173, this analysis showed rearrangement of Ig heavy chain and beta-TCR genes, unrearranged Ig light chain DNA, and expression of only an immature beta-TCR transcript. This line provides evidence for T-cell lineage involvement in Ph1 CML. One cell line without markers of any cell type, KCL-22, demonstrated rearranged, unexpressed Ig heavy chain genes, suggesting these cells are at the very earliest stages of lymphoid differentiation. These lines should provide valuable tools to dissect the molecular biology of differentiation in CML and in early lymphocytes.

  19. CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability

    Science.gov (United States)

    Inoue, Takeshi; Morita, Masahiro; Hijikata, Atsushi; Fukuda-Yuzawa, Yoko; Adachi, Shungo; Isono, Kyoichi; Ikawa, Tomokatsu; Kawamoto, Hiroshi; Koseki, Haruhiko; Natsume, Tohru; Fukao, Taro; Ohara, Osamu; Yamamoto, Tadashi

    2015-01-01

    The CCR4–NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre–B cell transition. CNOT3 regulated generation of germline transcripts in the VH region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4–NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA. PMID:26238124

  20. Carbon nanotubes film preparation on 3D structured silicon substrates by spray coating technique for application in solar cells

    Science.gov (United States)

    Xiang, Y.; Li, M.; Lin, C.; Liu, P.; Zhang, J.

    2014-11-01

    This paper firstly reports the preparation of carbon nanotubes (CNTs) film on silicon substrate of three-dimensional (3D) inverted pyramid structure (IPS) by spray coating. The effect of different substrate temperatures, spraying times and opening sizes on CNTs sidewall covering properties were investigated. The results show that the CNTs covering ratio of sidewall is much lower than that of flat surface and gradually decrease with depth. 40μm×40μm opening obtained the best sidewall covering by CNTs suspension of 40μg/ml at 120°C after 30min spraying so that the CNTs can reach the bottom of IPS and cover about 68.9% sidewall area. At last, it is demonstrated that the output power of the CNTs film-Si solar cell can be enhanced 5.7 times by this method compared to that of the plane structure.

  1. Heat- and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold.

    Science.gov (United States)

    Navarra, Giovanna; Peres, Chiara; Contardi, Marco; Picone, Pasquale; San Biagio, Pier Luigi; Di Carlo, Marta; Giacomazza, Daniela; Militello, Valeria

    2016-09-15

    Aggregation and gelation of globular proteins can be an advantage to generate new forms of nanoscale biomaterials based on the fibrillar architecture. Here, we report results obtained by exploiting the proteins' natural tendency to self-organize in 3D network, for the production of new material based on BSA for medical application. In particular, at five different pH values the conformational and structural changes of the BSA during all the steps of the thermal aggregation and gelation have been analyzed by FTIR spectroscopy. The macroscopic mechanical properties of these hydrogels have been obtained by rheological measurements. The microscopic structure of the gels have been studied by AFM and SEM images to have a picture of their different spatial arrangement. Finally, the use of the BSA hydrogels as scaffold has been tested in two different cell cultures.

  2. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution.

    Science.gov (United States)

    Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S; Edwards, Thayne L; James, Conrad D; Lidke, Keith A

    2016-06-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

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

    Directory of Open Access Journals (Sweden)

    Machado-Santelli Glaucia M

    2008-06-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  5. Nanoengineered implant as a new platform for regenerative nanomedicine using 3D well-organized human cell spheroids

    Directory of Open Access Journals (Sweden)

    Keller L

    2017-01-01

    Full Text Available Laetitia Keller,1,2,* Ysia Idoux-Gillet,1,2,* Quentin Wagner,1,2,* Sandy Eap,1,2,* David Brasse,3 Pascale Schwinté,1,2 Manuel Arruebo,4 Nadia Benkirane-Jessel1,2 1INSERM (French National Institute of Health and Medical Research, “Osteoarticular and Dental Regenerative Nanomedicine” Laboratory, UMR 1109, Faculté de Médecine, FMTS, 2University of Strasbourg, Faculté de Chirurgie Dentaire, 3CNRS (Centre National de la Recherche Scientifique, UMR 7178, IPHC (Hubert Curien Multidisciplinary Institute, Strasbourg, France; 4Department of Chemical Engineering, INA (Aragon Nanoscience Institute, University of Zaragoza, Zaragoza, Spain *These authors contributed equally to this work Abstract: In tissue engineering, it is still rare today to see clinically transferable strategies for tissue-engineered graft production that conclusively offer better tissue regeneration than the already existing technologies, decreased recovery times, and less risk of complications. Here a novel tissue-engineering concept is presented for the production of living bone implants combining 1 a nanofibrous and microporous implant as cell colonization matrix and 2 3D bone cell spheroids. This combination, double 3D implants, shows clinical relevant thicknesses for the treatment of an early stage of bone lesions before the need of bone substitutes. The strategy presented here shows a complete closure of a defect in nude mice calvaria after only 31 days. As a novel strategy for bone regenerative nanomedicine, it holds great promises to enhance the therapeutic efficacy of living bone implants. Keywords: bioengineering, implants, osteoblasts, matrix mineralization, microtissues

  6. Analysis of Chemotherapeutic Drug Delivery at the Single Cell Level Using 3D-MSI-TOF-SIMS

    Science.gov (United States)

    Vanbellingen, Quentin P.; Castellanos, Anthony; Rodriguez-Silva, Monica; Paudel, Iru; Chambers, Jeremy W.; Fernandez-Lima, Francisco A.

    2016-12-01

    In this work, we show the advantages of label-free, tridimensional mass spectrometry imaging using dual beam analysis (25 keV Bi3 +) and depth profiling (20 keV with a distribution centered at Ar1500 +) coupled to time of flight secondary ion mass spectrometry (3D-MSI-TOF-SIMS) for the study of A-172 human glioblastoma cell line treated with B-cell lymphoma 2 (Bcl-2) inhibitor ABT-737. The high spatial ( 250 nm) and high mass resolution (m/Δm 10,000) of TOF-SIMS permitted the localization and identification of the intact, unlabeled drug molecular ion ( m/z 811.26 C42H44ClN6O5S2 - [M - H]-) as well as characteristic fragment ions. We propose a novel approach based on the inspection of the drug secondary ion yield, which showed a good correlation with the drug concentration during cell treatment at therapeutic dosages (0-200 μM with 4 h incubation). Chemical maps using endogenous molecular markers showed that the ABT-737 is mainly localized in subsurface regions and absent in the nucleus. A semiquantitative workflow is proposed to account for the biological cell diversity based on the spatial distribution of endogenous molecular markers (e.g., nuclei and cytoplasm) and secondary ion confirmation based on the ratio of drug-specific fragments to molecular ion as a function of the therapeutic dosage.

  7. Implementation and Performance Modeling of Deterministic Particle Transport (Sweep3D on the IBM Cell/B.E.

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    Olaf Lubeck

    2009-01-01

    Full Text Available The IBM Cell Broadband Engine (BE is a novel multi-core chip with the potential for the demanding floating point performance that is required for high-fidelity scientific simulations. However, data movement within the chip can be a major challenge to realizing the benefits of the peak floating point rates. In this paper, we present the results of implementing Sweep3D on the Cell/B.E. using an intra-chip message passing model that minimizes data movement. We compare the advantages/disadvantages of this programming model with a previous implementation using a master–worker threading strategy. We apply a previously validated micro-architecture performance model for the application executing on the Cell/B.E. (based on our previous work in Monte Carlo performance models, that predicts overall CPI (cycles per instruction, and gives a detailed breakdown of processor stalls. Finally, we use the micro-architecture model to assess the performance of future design parameters for the Cell/B.E. micro-architecture. The methodologies and results have broader implications that extend to multi-core architectures.

  8. The Synergistic Effects of Matrix Stiffness and Composition on the Response of Chondroprogenitor Cells in a 3D Precondensation Microenvironment.

    Science.gov (United States)

    Carrion, Bita; Souzanchi, Mohammad F; Wang, Victor T; Tiruchinapally, Gopinath; Shikanov, Ariella; Putnam, Andrew J; Coleman, Rhima M

    2016-05-01

    Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Nanostructured p-type CZTS thin films prepared by a facile solution process for 3D p-n junction solar cells.

    Science.gov (United States)

    Park, Si-Nae; Sung, Shi-Joon; Sim, Jun-Hyoung; Yang, Kee-Jeong; Hwang, Dae-Kue; Kim, JunHo; Kim, Gee Yeong; Jo, William; Kim, Dae-Hwan; Kang, Jin-Kyu

    2015-07-07

    Nanoporous p-type semiconductor thin films prepared by a simple solution-based process with appropriate thermal treatment and three-dimensional (3D) p-n junction solar cells fabricated by depositing n-type semiconductor layers onto the nanoporous p-type thin films show considerable photovoltaic performance compared with conventional thin film p-n junction solar cells. Spin-coated p-type Cu2ZnSnS4 (CZTS) thin films prepared using metal chlorides and thiourea show unique nanoporous thin film morphology, which is composed of a cluster of CZTS nanograins of 50-500 nm, and the obvious 3D p-n junction structure is fabricated by the deposition of n-type CdS on the nanoporous CZTS thin films by chemical bath deposition. The photovoltaic properties of 3D p-n junction CZTS solar cells are predominantly affected by the scale of CZTS nanograins, which is easily controlled by the sulfurization temperature of CZTS precursor films. The scale of CZTS nanograins determines the minority carrier transportation within the 3D p-n junction between CZTS and CdS, which are closely related with the photocurrent of series resistance of 3D p-n junction solar cells. 3D p-n junction CZTS solar cells with nanograins below 100 nm show power conversion efficiency of 5.02%, which is comparable with conventional CZTS thin film solar cells.

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

    Science.gov (United States)

    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 448–5804 Pa modestly regulated SMC cytoskeletal assembly in 3-D, with spread cells in stiff matrices having a slightly higher degree of F-actin bundling after prolonged culture. However, vinculin expression in all 3-D conditions was qualitatively low and was not assembled into the classic focal adhesions typically seen in 2-D cultures. Given the evidence that RhoA-mediated cytoskeletal contractility represents a critical node in mechanosensing, we molecularly upregulated contractility by inducing SMCs to express constitutively active RhoA. In these cells, F-actin bundling and total vinculin expression increased, and focal adhesion-like structures began to emerge, consistent with RhoA’s mechanism of action cells cultured on 2-D substrates. Furthermore, SMC proliferation in 3-D did not depend significantly on matrix stiffness, and was reduced by constitutive activation of RhoA irrespective of ECM mechanical properties. Conversely, the expression of contractile markers globally increased with constitutive RhoA activation and depended on 3-D matrix stiffness only in cells with heightened RhoA activity. Combined, these data suggest the synergistic effects of ECM mechanics and RhoA activity on SMC phenotype in 3-D are distinct from those in 2-D, and highlight the importance of studying the mechanical role of cell-matrix interactions in tunable 3-D environments. PMID:18342366

  12. A computer-assisted 3D model for analyzing the aggregation of tumorigenic cells reveals specialized behaviors and unique cell types that facilitate aggregate coalescence.

    Directory of Open Access Journals (Sweden)

    Amanda Scherer

    Full Text Available We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity, and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named "facilitators" and "probes." A third cell type, the "dervish", is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.

  13. Resveratrol, Acetyl-Resveratrol, and Polydatin Exhibit Antigrowth Activity against 3D Cell Aggregates of the SKOV-3 and OVCAR-8 Ovarian Cancer Cell Lines

    OpenAIRE

    Hogg, Simon J.; Kenny Chitcholtan; Wafaa Hassan; Peter H. Sykes; Ashley Garrill

    2015-01-01

    Resveratrol has aroused significant scientific interest as it has been claimed that it exhibits a spectrum of health benefits. These include effects as an anti-inflammatory and an antitumour compound. The purpose of this study was to investigate and compare any potential antigrowth effects of resveratrol and two of its derivatives, acetyl-resveratrol and polydatin, on 3D cell aggregates of the EGFR/Her-2 positive and negative ovarian cancer cell lines SKOV-3 and OVCAR-8, respectively. Results...

  14. Strategy to Achieve Highly Porous/Biocompatible Macroscale Cell Blocks, Using a Collagen/Genipin-bioink and an Optimal 3D Printing Process.

    Science.gov (United States)

    Kim, Yong Bok; Lee, Hyeongjin; Kim, Geun Hyung

    2016-11-30

    Recently, a three-dimensional (3D) bioprinting process for obtaining a cell-laden structure has been widely applied because of its ability to fabricate biomimetic complex structures embedded with and without cells. To successfully obtain a cell-laden porous block, the cell-delivering vehicle, bioink, is one of the significant factors. Until now, various biocompatible hydrogels (synthetic and natural biopolymers) have been utilized in the cell-printing process, but a bioink satisfying both biocompatibility and print-ability requirements to achieve a porous structure with reasonable mechanical strength has not been issued. Here, we propose a printing strategy with optimal conditions including a safe cross-linking procedure for obtaining a 3D porous cell block composed of a biocompatible collagen-bioink and genipin, a cross-linking agent. To obtain the optimal processing conditions, we modified the 3D printing machine and selected an optimal cross-linking condition (∼1 mM and 1 h) of genipin solution. To show the feasibility of the process, 3D pore-interconnected cell-laden constructs were manufactured using osteoblast-like cells (MG63) and human adipose stem cells (hASCs). Under these processing conditions, a macroscale 3D collagen-based cell block of 21 × 21 × 12 mm(3) and over 95% cell viability was obtained. In vitro biological testing of the cell-laden 3D porous structure showed that the embedded cells were sufficiently viable, and their proliferation was significantly higher; the cells also exhibited increased osteogenic activities compared to the conventional alginate-based bioink (control). The results indicated the fabrication process using the collagen-bioink would be an innovative platform to design highly biocompatible and mechanically stable cell blocks.

  15. 3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

    Science.gov (United States)

    Malenovská, Hana

    2016-02-01

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

  16. Prediction of conformational B-cell epitopes from 3D structures by random forests with a distance-based feature

    Directory of Open Access Journals (Sweden)

    Zou Hua

    2011-08-01

    Full Text Available Abstract Background Antigen-antibody interactions are key events in immune system, which provide important clues to the immune processes and responses. In Antigen-antibody interactions, the specific sites on the antigens that are directly bound by the B-cell produced antibodies are well known as B-cell epitopes. The identification of epitopes is a hot topic in bioinformatics because of their potential use in the epitope-based drug design. Although most B-cell epitopes are discontinuous (or conformational, insufficient effort has been put into the conformational epitope prediction, and the performance of existing methods is far from satisfaction. Results In order to develop the high-accuracy model, we focus on some possible aspects concerning the prediction performance, including the impact of interior residues, different contributions of adjacent residues, and the imbalanced data which contain much more non-epitope residues than epitope residues. In order to address above issues, we take following strategies. Firstly, a concept of 'thick surface patch' instead of 'surface patch' is introduced to describe the local spatial context of each surface residue, which considers the impact of interior residue. The comparison between the thick surface patch and the surface patch shows that interior residues contribute to the recognition of epitopes. Secondly, statistical significance of the distance distribution difference between non-epitope patches and epitope patches is observed, thus an adjacent residue distance feature is presented, which reflects the unequal contributions of adjacent residues to the location of binding sites. Thirdly, a bootstrapping and voting procedure is adopted to deal with the imbalanced dataset. Based on the above ideas, we propose a new method to identify the B-cell conformational epitopes from 3D structures by combining conventional features and the proposed feature, and the random forest (RF algorithm is used as the

  17. Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

    Science.gov (United States)

    Whetsell, M; Mosley, R L; Whetsell, L; Schaefer, F V; Miller, K S; Klein, J R

    1991-12-01

    The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.

  18. Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

    Science.gov (United States)

    Zhang, W; Kong, C W; Tong, M H; Chooi, W H; Huang, N; Li, R A; Chan, B P

    2017-02-01

    Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study.

  19. Cytotoxic responses of carnosic acid and doxorubicin on breast cancer cells in butterfly-shaped microchips in comparison to 2D and 3D culture.

    Science.gov (United States)

    Yildiz-Ozturk, Ece; Gulce-Iz, Sultan; Anil, Muge; Yesil-Celiktas, Ozlem

    2017-04-01

    Two dimensional (2D) cell culture systems lack the ability to mimic in vivo conditions resulting in limitations for preclinical cell-based drug and toxicity screening assays and modelling tumor biology. Alternatively, 3D cell culture systems mimic the specificity of native tissue with better physiological integrity. In this regard, microfluidic chips have gained wide applicability for in vitro 3D cancer cell studies. The aim of this research was to develop a 3D biomimetic model comprising culture of breast cancer cells in butterfly-shaped microchip to determine the cytotoxicity of carnosic acid and doxorubicin on both estrogen dependent (MCF-7) and independent (MDA-MB231) breast cancer cells along with healthy mammary epithelial cells (MCF-10A) in 2D, 3D Matrigel™ and butterfly-shaped microchip environment. According to the developed mimetic model, carnosic acid exhibited a higher cytotoxicity towards MDA-MB 231, while doxorubicin was more effective against MCF-7. Although the cell viabilities were higher in comparison to 2D and 3D cell culture systems, the responses of the investigated molecules were different in the microchips based on the molecular weight and structural complexity indicating the importance of biomimicry in a physiologically relevant matrix.

  20. 3D Animation Essentials

    CERN Document Server

    Beane, Andy

    2012-01-01

    The essential fundamentals of 3D animation for aspiring 3D artists 3D is everywhere--video games, movie and television special effects, mobile devices, etc. Many aspiring artists and animators have grown up with 3D and computers, and naturally gravitate to this field as their area of interest. Bringing a blend of studio and classroom experience to offer you thorough coverage of the 3D animation industry, this must-have book shows you what it takes to create compelling and realistic 3D imagery. Serves as the first step to understanding the language of 3D and computer graphics (CG)Covers 3D anim

  1. 3D video

    CERN Document Server

    Lucas, Laurent; Loscos, Céline

    2013-01-01

    While 3D vision has existed for many years, the use of 3D cameras and video-based modeling by the film industry has induced an explosion of interest for 3D acquisition technology, 3D content and 3D displays. As such, 3D video has become one of the new technology trends of this century.The chapters in this book cover a large spectrum of areas connected to 3D video, which are presented both theoretically and technologically, while taking into account both physiological and perceptual aspects. Stepping away from traditional 3D vision, the authors, all currently involved in these areas, provide th

  2. Consistency of VDJ Rearrangement and Substitution Parameters Enables Accurate B Cell Receptor Sequence Annotation.

    Directory of Open Access Journals (Sweden)

    Duncan K Ralph

    2016-01-01

    Full Text Available VDJ rearrangement and somatic hypermutation work together to produce antibody-coding B cell receptor (BCR sequences for a remarkable diversity of antigens. It is now possible to sequence these BCRs in high throughput; analysis of these sequences is bringing new insight into how antibodies develop, in particular for broadly-neutralizing antibodies against HIV and influenza. A fundamental step in such sequence analysis is to annotate each base as coming from a specific one of the V, D, or J genes, or from an N-addition (a.k.a. non-templated insertion. Previous work has used simple parametric distributions to model transitions from state to state in a hidden Markov model (HMM of VDJ recombination, and assumed that mutations occur via the same process across sites. However, codon frame and other effects have been observed to violate these parametric assumptions for such coding sequences, suggesting that a non-parametric approach to modeling the recombination process could be useful. In our paper, we find that indeed large modern data sets suggest a model using parameter-rich per-allele categorical distributions for HMM transition probabilities and per-allele-per-position mutation probabilities, and that using such a model for inference leads to significantly improved results. We present an accurate and efficient BCR sequence annotation software package using a novel HMM "factorization" strategy. This package, called partis (https://github.com/psathyrella/partis/, is built on a new general-purpose HMM compiler that can perform efficient inference given a simple text description of an HMM.

  3. Potential Synaptic Connectivity of Different Neurons onto Pyramidal Cells in a 3D Reconstruction of the Rat Hippocampus

    Directory of Open Access Journals (Sweden)

    Deepak eRopireddy

    2011-07-01

    Full Text Available Most existing connectomic data and ongoing efforts focus either on individual synapses (e.g. with electron microscopy or on regional connectivity (tract tracing. An individual pyramidal cell extends thousands of synapses over macroscopic distances (~cm. The contrasting requirements of high resolution and large field of view make it too challenging to acquire the entire synaptic connectivity for even a single typical cortical neuron. Light microscopy can image whole neuronal arbors and resolve dendritic branches. Analyzing connectivity in terms of close spatial appositions between axons and dendrites could thus bridge the opposite scales, from synaptic level to whole systems. In the mammalian cortex, structural plasticity of spines and boutons makes these ‘potential synapses’ functionally relevant to learning capability and memory capacity. To date, however, potential synapses have only been mapped in the surrounding of a neuron and relative to its local orientation rather than in a system-level anatomical reference. Here we overcome this limitation by estimating the potential connectivity of different neurons embedded into a detailed 3D reconstruction of the rat hippocampus. Axonal and dendritic trees were oriented with respect to hippocampal cytoarchitecture according to longitudinal and transversal curvatures. We report the potential connectivity onto pyramidal cell dendrites from the axons of a dentate granule cell, three CA3 pyramidal cells, one CA2 pyramidal cell, and 13 CA3b interneurons. The numbers, densities, and distributions of potential synapses were analyzed in each sub-region (e.g. CA3 vs. CA1, layer (e.g. oriens vs. radiatum, and septo-temporal location (e.g. dorsal vs. ventral. The overall ratio between the numbers of actual and potential synapses was ~0.20 for the granule and CA3 pyramidal cells. All potential connectivity patterns are strikingly dependent on the anatomical location of both pre-synaptic and post

  4. Study of 11 September 2004 hailstorm event using radar identification of 2-D systems and 3-D cells

    Directory of Open Access Journals (Sweden)

    M. Ceperuelo

    2006-01-01

    Full Text Available The most important hail event recorded in the region of the Ebro Valley (NE Spain in 2004 was the 11 September episode. Large hailstones (some of them with a diameter of over 30 mm caused important damages in agriculture and properties. The hail event affected an area of 3848 ha and was caused by several multicellular systems. The aim of this paper is the analysis of the associated convective structures using the meteorological radar as well as the MM5 mesoscale model, thermodynamic data and a hailpad network. To achieve this end, the new hailstorm analysis tool RHAP (Rainfall events and Hailstorms Analysis Program has been applied. It identifies tracks and characterises precipitation systems and convective cells, taking into account 2-D and 3-D structures. The event has also been studied with the TITAN software (Thunderstorm Identification, Tracking, Analysis and Nowcasting in an attempt to compare both methods. Results show that the episode had a strong convective activity with CAPE values over 4000 J/kg and with hail-forming cells characterised by VIL (Vertical Integrated Liguid exceeding 40 kg/m2, VILD (VIL density over 4 g/m3, HP (Hail Probability of 100% and SHP (Severe Hail Probability of over 75%. The hail cells evolved into multicellular systems that lasted between 70 and 90 min. Finally, the comparison of RHAP and TITAN has shown significant correlations between methods.

  5. Aggregatibacter actinomycetemcomitans Omp29 is associated with bacterial entry to gingival epithelial cells by F-actin rearrangement.

    Directory of Open Access Journals (Sweden)

    Mikihito Kajiya

    Full Text Available The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29, a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+/OmpA(- E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9 than Omp29(-/OmpA(- E. coli. While the entry of Aa and Omp29(+/OmpA(- E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK, a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin and Rho GTPases inhibitor (EDIN, both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

  6. A nano-microstructured artificial-hair-cell-type sensor based on topologically graded 3D carbon nanotube bundles

    Science.gov (United States)

    Yilmazoglu, O.; Yadav, S.; Cicek, D.; Schneider, J. J.

    2016-09-01

    A design for a unique artificial-hair-cell-type sensor (AHCTS) based entirely on 3D-structured, vertically aligned carbon nanotube (CNT) bundles is introduced. Standard microfabrication techniques were used for the straightforward micro-nano integration of vertically aligned carbon nanotube arrays composed of low-layer multi-walled CNTs (two to six layers). The mechanical properties of the carbon nanotube bundles were intensively characterized with regard to various substrates and CNT morphology, e.g. bundle height. The CNT bundles display excellent flexibility and mechanical stability for lateral bending, showing high tear resistance. The integrated 3D CNT sensor can detect three-dimensional forces using the deflection or compression of a central CNT bundle which changes the contact resistance to the shorter neighboring bundles. The complete sensor system can be fabricated using a single chemical vapor deposition (CVD) process step. Moreover, sophisticated external contacts to the surroundings are not necessary for signal detection. No additional sensors or external bias for signal detection are required. This simplifies the miniaturization and the integration of these nanostructures for future microsystem set-ups. The new nanostructured sensor system exhibits an average sensitivity of 2100 ppm in the linear regime with the relative resistance change per micron (ppm μm-1) of the individual CNT bundle tip deflection. Furthermore, experiments have shown highly sensitive piezoresistive behavior with an electrical resistance decrease of up to ˜11% at 50 μm mechanical deflection. The detection sensitivity is as low as 1 μm of deflection, and thus highly comparable with the tactile hair sensors of insects, having typical thresholds on the order of 30-50 μm. The AHCTS can easily be adapted and applied as a flow, tactile or acceleration sensor as well as a vibration sensor. Potential applications of the latter might come up in artificial cochlear systems. In

  7. A nano-microstructured artificial-hair-cell-type sensor based on topologically graded 3D carbon nanotube bundles.

    Science.gov (United States)

    Yilmazoglu, O; Yadav, S; Cicek, D; Schneider, J J

    2016-09-09

    A design for a unique artificial-hair-cell-type sensor (AHCTS) based entirely on 3D-structured, vertically aligned carbon nanotube (CNT) bundles is introduced. Standard microfabrication techniques were used for the straightforward micro-nano integration of vertically aligned carbon nanotube arrays composed of low-layer multi-walled CNTs (two to six layers). The mechanical properties of the carbon nanotube bundles were intensively characterized with regard to various substrates and CNT morphology, e.g. bundle height. The CNT bundles display excellent flexibility and mechanical stability for lateral bending, showing high tear resistance. The integrated 3D CNT sensor can detect three-dimensional forces using the deflection or compression of a central CNT bundle which changes the contact resistance to the shorter neighboring bundles. The complete sensor system can be fabricated using a single chemical vapor deposition (CVD) process step. Moreover, sophisticated external contacts to the surroundings are not necessary for signal detection. No additional sensors or external bias for signal detection are required. This simplifies the miniaturization and the integration of these nanostructures for future microsystem set-ups. The new nanostructured sensor system exhibits an average sensitivity of 2100 ppm in the linear regime with the relative resistance change per micron (ppm μm(-1)) of the individual CNT bundle tip deflection. Furthermore, experiments have shown highly sensitive piezoresistive behavior with an electrical resistance decrease of up to ∼11% at 50 μm mechanical deflection. The detection sensitivity is as low as 1 μm of deflection, and thus highly comparable with the tactile hair sensors of insects, having typical thresholds on the order of 30-50 μm. The AHCTS can easily be adapted and applied as a flow, tactile or acceleration sensor as well as a vibration sensor. Potential applications of the latter might come up in artificial cochlear systems. In

  8. Bioprinting of 3D hydrogels.

    Science.gov (United States)

    Stanton, M M; Samitier, J; Sánchez, S

    2015-08-07

    Three-dimensional (3D) bioprinting has recently emerged as an extension of 3D material printing, by using biocompatible or cellular components to build structures in an additive, layer-by-layer methodology for encapsulation and culture of cells. These 3D systems allow for cell culture in a suspension for formation of highly organized tissue or controlled spatial orientation of cell environments. The in vitro 3D cellular environments simulate the complexity of an in vivo environment and natural extracellular matrices (ECM). This paper will focus on bioprinting utilizing hydrogels as 3D scaffolds. Hydrogels are advantageous for cell culture as they are highly permeable to cell culture media, nutrients, and waste products generated during metabolic cell processes. They have the ability to be fabricated in customized shapes with various material properties with dimensions at the micron scale. 3D hydrogels are a reliable method for biocompatible 3D printing and have applications in tissue engineering, drug screening, and organ on a chip models.

  9. Metabonomic study on the antitumor effect of flavonoid derivative 3d in HepG2 cells and its action mechanism.

    Science.gov (United States)

    Gao, Dan; Jin, Feng; Liu, Hongxia; Wang, Yini; Jiang, Yuyang

    2014-01-01

    A novel flavonid derivate, 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chlorophenyl)urea (3d) synthesized in our lab possesses potent antitumor activity against HepG2 cells. Our previous studies on pharmacological mechanism of 3d mostly focused on cell and gene levels, little is about its metabolomics study. Herein, an ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) based metabolomics approach was established to investigate the antitumor effect of 3d on HepG2 cells and its action mechanism. Q-TOF MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. Comparing 3d-treated HepG2 cells with vehicle control (dimethyl sulfoxide), 32 distinct metabolites involved in glutathione metabolism, glycerophospholipid metabolism, cysteine and methionine metabolism, fatty acid metabolism, and phenylalanine metabolism. The reduced level of glutathione (GSH) and decreased ratio of reduced/oxidized glutathione (GSH/GSSG) in 3d-treated cells indicated the increased oxidative stress after 3d treatment. The significant decrease of phosphatidylcholine (PC) levels and increase of lysophosphatidylcholine (LPC) levels suggested alterations in lipid composition which were causally related to decline in mitochondrial function. Depletion of carnitine and increase of long chain carnitines and fatty acids reflected decline in fatty acid metabolism. The further biological experiments including ROS and MMP measurements confirmed the above probabilities presumed from metabolomic results. Our findings suggested that 3d caused the perturbation of multiple cellular pathways. The increased oxidative stress and the resulting mitochondrial dysfunction resulted in the antiproliferative effect of 3d. The UPLC/Q-TOF MS based metabolomics approach provides new insights into the mechanistic studies of new compounds that distinct from traditional biological studies.

  10. The effects of 3D channel geometry on CTC passing pressure--towards deformability-based cancer cell separation.

    Science.gov (United States)

    Zhang, Zhifeng; Xu, Jie; Hong, Bin; Chen, Xiaolin

    2014-07-21

    Various lab on a chip devices have been developed recently to detect and separate circulating tumour cells (CTCs) for early stage cancer detection. Because CTCs are extremely rare in the blood, next generation CTC microfilters aim at significant improvement in both efficiency and throughput. CTC microfilters based on cell deformability seem to be a promising direction. In the present research, we study a CTC passing event through a micro-filtering channel with various 3D geometries. The pressure signatures for different types of cells passing through different channels are characterized numerically. Specifically, five kinds of cross-sections, circular, square, triangular and two kinds of rectangular channels with aspect ratios of 2 and 5, are studied in this work. The total pressures for cells passing through the channels are calculated and reveal different behaviour from what is predicted by the static surface tension model. Among all five cross-sections studied, the circular cross-section features the highest critical pressure and thus is most suitable for high efficiency CTC separation. The square filtering channel provides the second largest critical pressure, and the triangular cross-section provides the least critical pressure among these three cross-sections. All these three cross-sections are better than the rectangular channels with aspect ratios of 2 and 5. For the rectangular channel, a high aspect ratio channel may lead to cell splitting at high speed, which will result in a periodic pressure signature. Our findings will provide valuable information for the design of next generation CTC microfilters.

  11. Laser-based microdissection of single cells from tissue sections and PCR analysis of rearranged immunoglobulin genes from isolated normal and malignant human B cells.

    Science.gov (United States)

    Küppers, Ralf; Schneider, Markus; Hansmann, Martin-Leo

    2013-01-01

    Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene family-specific primers recognizing nearly all V gene segments together with primers for the J gene segments. By sequence analysis of V genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

  12. Replication stalling by catalytically impaired Twinkle induces mitochondrial DNA rearrangements in cultured cells

    NARCIS (Netherlands)

    Pohjoismaki, J.L.; Goffart, S.; Spelbrink, J.N.

    2011-01-01

    Pathological mitochondrial DNA (mtDNA) rearrangements have been proposed to result from repair of double-strand breaks caused by blockage of mitochondrial DNA (mtDNA) replication. As mtDNA deletions are seen only in post-mitotic tissues, it has been suggested that they are selected out in actively d

  13. Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division.

    Science.gov (United States)

    Bala, Shashi; Kumar, Ajay; Soni, Shivani; Sinha, Sudha; Hanspal, Manjit

    2006-04-21

    Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

  14. A New Crank Arm-Based Load Cell for the 3D Analysis of the Force Applied by a Cyclist

    Directory of Open Access Journals (Sweden)

    Alexandre Balbinot

    2014-12-01

    Full Text Available This report describes a new crank arm-based force platform designed to evaluate the three-dimensional force applied to the pedals by cyclists in real conditions. The force platform was designed to be fitted on a conventional competition bicycle crankset while data is transmitted wirelessly through a BluetoothTM module and also stored on a SD card. A 3D solid model is created in the SolidWorks (Dassault Systèmes SOLIDWORKS Corp. to analyze the static and dynamic characteristics of the crank arm by using the finite elements technique. Each crankset arm is used as a load cell based on strain gauges configured as three Wheatstone bridges. The signals are conditioned on a printed circuit board attached directly to the structure. The load cell showed a maximum nonlinearity error between 0.36% and 0.61% and a maximum uncertainty of 2.3% referred to the sensitivity of each channel. A roller trainer equipped with an optical encoder was also developed, allowing the measurement of the wheel’s instantaneous velocity.

  15. Scalable, incremental learning with MapReduce parallelization for cell detection in high-resolution 3D microscopy data

    KAUST Repository

    Sung, Chul

    2013-08-01

    Accurate estimation of neuronal count and distribution is central to the understanding of the organization and layout of cortical maps in the brain, and changes in the cell population induced by brain disorders. High-throughput 3D microscopy techniques such as Knife-Edge Scanning Microscopy (KESM) are enabling whole-brain survey of neuronal distributions. Data from such techniques pose serious challenges to quantitative analysis due to the massive, growing, and sparsely labeled nature of the data. In this paper, we present a scalable, incremental learning algorithm for cell body detection that can address these issues. Our algorithm is computationally efficient (linear mapping, non-iterative) and does not require retraining (unlike gradient-based approaches) or retention of old raw data (unlike instance-based learning). We tested our algorithm on our rat brain Nissl data set, showing superior performance compared to an artificial neural network-based benchmark, and also demonstrated robust performance in a scenario where the data set is rapidly growing in size. Our algorithm is also highly parallelizable due to its incremental nature, and we demonstrated this empirically using a MapReduce-based implementation of the algorithm. We expect our scalable, incremental learning approach to be widely applicable to medical imaging domains where there is a constant flux of new data. © 2013 IEEE.

  16. Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

    Science.gov (United States)

    Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

    2000-04-01

    Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

  17. COUPLED SIMULATION OF 3D ELECTRO-MAGNETO-FLOW FIELD IN HALL-HEROULT CELLS USING FINITE ELEMENT METHOD

    Institute of Scientific and Technical Information of China (English)

    J. Li; W. Liu; Y.Q. Lai; Q.Y. Li; Y.X. Liu

    2006-01-01

    Two full 3D steady mathematical models are developed by finite element method (FEM) to calculate coupled physics fields: the electro-magnetic model is built and solved first and so is the fluid motion model with the acquired electromagnetic force as source body forces in Navier-Stokes equations. Effects caused by the ferromagnetic shell, busbar system around, and open boundary problem as well as inside induced current were considered in terms of the magnetic field. Furthermore, a new modeling method is found to set up solid models and then mesh them entirely with so-called structuralized grids, namely hex-mesh. Examples of 75kA prebaked cell with two kinds of busbar arrangements are presented. Results agree with those disclosed in the literature and confirm that the coupled simulation is valid. It is also concluded that the usage of these models facilitates the consistent analysis of the electric field to magnetic field and then flow motion to the greater extent, local distributions of current density and magnetic flux density are very much dependent on the cell structure, the steel shell is a shield to reduce the magnetic field and flow pattern is two dimensional in the main body of the metal pad.

  18. A new crank arm-based load cell for the 3D analysis of the force applied by a cyclist.

    Science.gov (United States)

    Balbinot, Alexandre; Milani, Cleiton; Nascimento, Jussan da Silva Bahia

    2014-12-03

    This report describes a new crank arm-based force platform designed to evaluate the three-dimensional force applied to the pedals by cyclists in real conditions. The force platform was designed to be fitted on a conventional competition bicycle crankset while data is transmitted wirelessly through a Bluetooth™ module and also stored on a SD card. A 3D solid model is created in the SolidWorks (Dassault Systèmes SOLIDWORKS Corp.) to analyze the static and dynamic characteristics of the crank arm by using the finite elements technique. Each crankset arm is used as a load cell based on strain gauges configured as three Wheatstone bridges. The signals are conditioned on a printed circuit board attached directly to the structure. The load cell showed a maximum nonlinearity error between 0.36% and 0.61% and a maximum uncertainty of 2.3% referred to the sensitivity of each channel. A roller trainer equipped with an optical encoder was also developed, allowing the measurement of the wheel's instantaneous velocity.

  19. Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

    Science.gov (United States)

    Lance, Amanda; Yang, Chih-Chao; Swamydas, Muthulekha; Dean, Delphine; Deitch, Sandy; Burg, Karen J L; Dréau, Didier

    2016-01-01

    The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ß-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Clear cell myoepithelial carcinoma of salivary glands showing EWSR1 rearrangement: molecular analysis of 94 salivary gland carcinomas with prominent clear cell component.

    Science.gov (United States)

    Skálová, Alena; Weinreb, Ilan; Hyrcza, Martin; Simpson, Roderick H W; Laco, Jan; Agaimy, Abbas; Vazmitel, Marina; Majewska, Hanna; Vanecek, Tomas; Talarčik, Peter; Manajlovic, Spomenka; Losito, Simona N; Šteiner, Petr; Klimkova, Adela; Michal, Michal

    2015-03-01

    This study examines the presence of the EWSR1 rearrangement in a variety of clear cell salivary gland carcinomas with myoepithelial differentiation. A total of 94 salivary gland carcinomas with a prominent clear cell component included 51 cases of clear cell myoepithelial carcinomas de novo (CCMC), 21 cases of CCMCs ex pleomorphic adenoma (CCMCexPA), 11 cases of epithelial-myoepithelial carcinoma (EMC), 6 cases of EMC with solid clear cell overgrowth, and 5 cases of hyalinizing clear cell carcinoma of minor salivary glands. In addition, 10 cases of myoepithelial carcinomas devoid of clear cell change and 12 cases of benign myoepithelioma were included as well. All the tumors in this spectrum were reviewed, reclassified, and tested by fluorescence in situ hybridization (FISH) for the EWSR1 rearrangement using the Probe Vysis EWSR1 Break Apart FISH Probe Kit. The EWSR1 rearrangement was detected in 20 of 51 (39%) cases of CCMC, in 5 of 21 (24%) cases of CCMCexPA, in 1 of 11 (9%) cases of EMC, and in 4 of 5 (80%) cases of hyalinizing clear cell carcinoma. The 25 EWSR1-rearranged CCMCs and CCMCexPAs shared similar histomorphology. They were arranged in nodules composed of compact nests of large polyhedral cells with abundant clear cytoplasm. Necrosis, areas of squamous metaplasia, and hyalinization were frequent features. Immunohistochemically, the tumors expressed p63 (96%), cytokeratin CK14 (96%), and S100 protein (88%). MIB1 index varied from 10% to 100%, with most cases in the 20% to 40% range. Clinical follow-up information was available in 21 cases (84%) and ranged from 3 months to 15 years (mean 5.2 y); 4 patients were lost to follow-up. Ten patients are alive with no evidence of recurrent or metastatic disease in the follow-up period from 3 months to 15 years (mean 5 y), 3 patients are alive with recurrent and metastatic disease, and 8 died of disseminated cancer 9 months to 16 years after diagnosis (mean 6 y). Lymph node metastasis appeared in 5 patients

  2. Glycerol-mediated nanostructure modification leading to improved transparency of porous polymeric scaffolds for high performance 3D cell imaging.

    Science.gov (United States)

    Zhao, Shan; Shen, Zhiyuan; Wang, Jingyu; Li, Xiaokang; Zeng, Yang; Wang, Bingjie; He, Yonghong; Du, Yanan

    2014-07-14

    Glycerol is among the most commonly used optical clearing agents for tissues clearance largely due to refractive index (RI) matching between glycerol and the submerged tissues. Here we applied glycerol as structure modifier at both macroscopic (as porogen) and nanoscopic (as nanostructure ameliorant) scales to fabricate transparent porous scaffolds made from poly(ethylene glycol) (PEG) as well as other widely used biomaterials (e.g., PLGA, PA, or gelatin), whose nanostructures, in the scale of light wavelength, dominantly improved the optical transmittance of the scaffolds even when immersed in RI mismatched medium (e.g., culture medium or water). We further exploited the clearing mechanisms based on Mie scattering theory, illustrating that conformational changes of polymer chains induced by solvent effects of glycerol enhanced the anisotropy (i.e., directional alignment) of the nanostructures, leading to reduced crystallinity and scattering of the resulted PEG scaffolds. Our findings represent the first and systematic demonstration with both experimental and theoretical evidence in effectively clearing porous polymeric scaffolds by mechanisms other than RI matching, which could tackle the limitations of current optical imaging of cells cultured within three-dimensional (3D) opaque porous scaffolds, such as poor visibility, low spatial resolution, and small penetration depth.

  3. Implementation of a 3D version of ponderomotive guiding center solver in particle-in-cell code OSIRIS

    Science.gov (United States)

    Helm, Anton; Vieira, Jorge; Silva, Luis; Fonseca, Ricardo

    2016-10-01

    Laser-driven accelerators gained an increased attention over the past decades. Typical modeling techniques for laser wakefield acceleration (LWFA) are based on particle-in-cell (PIC) simulations. PIC simulations, however, are very computationally expensive due to the disparity of the relevant scales ranging from the laser wavelength, in the micrometer range, to the acceleration length, currently beyond the ten centimeter range. To minimize the gap between these despair scales the ponderomotive guiding center (PGC) algorithm is a promising approach. By describing the evolution of the laser pulse envelope separately, only the scales larger than the plasma wavelength are required to be resolved in the PGC algorithm, leading to speedups in several orders of magnitude. Previous work was limited to two dimensions. Here we present the implementation of the 3D version of a PGC solver into the massively parallel, fully relativistic PIC code OSIRIS. We extended the solver to include periodic boundary conditions and parallelization in all spatial dimensions. We present benchmarks for distributed and shared memory parallelization. We also discuss the stability of the PGC solver.

  4. 3D particle-in-cell simulation of electron acceleration by Langmuir waves in an inhomogeneous plasma

    CERN Document Server

    Pechhacker, R

    2014-01-01

    A possible solution to the unexplained high intensity hard x-ray (HXR) emission observable during solar flares was investigated via 3D fully relativistic, electromagnetic particle-in-cell (PIC) simulations with realistic ion to electron mass ratio. A beam of accelerated electrons was injected into a magnetised, Maxwellian, homogeneous and inhomogeneous background plasma. The electron distribution function was unstable to the beam-plasma instability and was shown to generate Langmuir waves, while relaxing to plateau formation. In order to estimate the role of the background density gradient on an unbound (infinite spatial extent) beam, three different scenarios were investigated: a) a uniform density background; b) a weak density gradient, n_R/n_L=3; c) a strong gradient case, n_R/n_L=10, where n_R and n_L denote background electron densities on the left and right edges of the simulation box respectively. The strong gradient case produced the largest fraction of electrons beyond 15 v_th. Further, two cases (un...

  5. Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Esmaiel Jabbari

    Full Text Available The growth and expression of cancer stem cells (CSCs depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells' tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA hydrogel without the interference of other factors in the microenvironment.Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x106 cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers.The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 μm.The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells' tissue origin.

  6. What do Cells Really Look Like? An Inquiry into Students' Difficulties in Visualising a 3-D Biological Cell and Lessons for Pedagogy

    Science.gov (United States)

    Vijapurkar, Jyotsna; Kawalkar, Aisha; Nambiar, Priya

    2014-04-01

    In our explorations of students' concepts in an inquiry science classroom with grade 6 students from urban schools in India, we uncovered a variety of problems in their understanding of biological cells as structural and functional units of living organisms. In particular, we found not only that they visualised the cell as a two-dimensional (2-D) structure, instead of a closed three-dimensional (3-D) functional unit, but that they had a strong resistance to changing their 2-D conception to a 3-D one. Based on analyses of students' oral as well as written descriptions of cells in the classroom, and of models they made of the cell, we were able to identify the causes of students' difficulties in correctly visualising the cell. These insights helped us design a pedagogy involving guided discussions and activities that challenges students' 2-D conceptions of the cell. The activities entail very simple, low-cost, easily doable techniques to help students visualise the cell and to understand that it would not be able to function if its structure were 2-D. We also present the results of our investigations of conceptions of grade 7 students and biology undergraduates, revealing that the incorrect 2-D mental model can persist right up to the college level if it is not explicitly addressed. The classroom interactions described in this study illustrate how students' ideas can be probed and addressed in the classroom using pedagogical action research.

  7. Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.

    Science.gov (United States)

    Berdoz, J; Monath, T P; Kraehenbuhl, J P

    1995-04-01

    We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa/lambda sequences. Variable regions with intact coding sequences, including full-length leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments that do not need to be adapted for recombinant antibody expression, thus facilitating the generation of chimeric and isotype-switched immunoglobulins.

  8. Revealing the Functions of Tenascin-C in 3-D Breast Cancer Models Using Cell Biological and in Silico Approaches

    Science.gov (United States)

    2008-03-01

    renditions of mammary acini, which were then used to assess and quantify acinar topography and volume. Although TN-C increased acinar surface roughness...epithelial 3-D tissue structure and function. In essence, we devised an algorithm to quantify acinar surface topography and volume in 3-D cultures of...deficient mice, Nature 1995, 377:539-544 39 34. Matsuda A, Yoshiki A, Tagawa Y, Matsuda H, Kusakabe M: Corneal wound healing in tenascin knockout mouse

  9. Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies

    Science.gov (United States)

    Mouka, Aurélie; Izard, Vincent; Tachdjian, Gérard; Brisset, Sophie; Yates, Frank; Mayeur, Anne; Drévillon, Loïc; Jarray, Rafika; Leboulch, Philippe; Maouche-Chrétien, Leila; Tosca, Lucie

    2017-01-01

    Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development. PMID:28045072

  10. Novel 3D scaffold with enhanced physical and cell response properties for bone tissue regeneration, fabricated by patterned electrospinning/electrospraying.

    Science.gov (United States)

    Hejazi, Fatemeh; Mirzadeh, Hamid

    2016-09-01

    Developing three dimensional scaffolds mimicking the nanoscale structure of native extracellular matrix is a key parameter in tissue regeneration. In this study, we aimed to introduce a novel 3D structures composed of nanofibers (NF) and micro particles (MP) and compare their efficiency with 2D nanofibrous scaffold. The conventional nanofibrous PCL scaffolds are 2D mats fabricated by the electrospinning technique, whereas the NF/MP and patterned NF/MP PCL scaffolds are three dimensional structures fabricated by a modified electrospinning/electrospraying technique. The mentioned method was carried out by varying the electrospinning solution parameters and use of a metal mesh as the collector. Detailed fabrication process and morphological properties of the fabricated structures is discussed and porosity, pore size and PBS solution absorption value of the prepared structures are reported. Compared with the 2D structure, 3D scaffolds possessed enhanced porosity and pore size which led to the significant increase in their water uptake capacity. In vitro cell experiments were carried out on the prepared structures by the use of MG-63 osteosarcoma cell line. The fabricated 3D structures offered significantly increased cell attachment, spread and diffusion which were confirmed by SEM analysis. In vitro cytocompatibility assessed by MTT colorimetric assay indicated a continuous cell proliferation over 21 days on the innovative 3D structure, while on 2D mat cell proliferation stopped at early time points. Enhanced osteogenic differentiation of the seeded MG-63 cells on 3D scaffold was confirmed by the remarkable ALP activity together with increased and accelerated calcium deposition on this structure compared to 2D mat. Massive and well distributed bone minerals formed on patterned 3D structure were shown by EDX analysis. In comparison between NF/MP quasi-3D and Patterned NF/MP 3D scaffolds, patterned structures proceeded in all of the above properties. As such, the

  11. Clinicopathologic characteristics andtherapeutic responses ofChinese patients withnon-small cell lung cancer who harbor an anaplastic lymphoma kinase rearrangement

    Institute of Scientific and Technical Information of China (English)

    ShaFu; HaiYunWang; FangWang; MaYanHuang; LingDeng; XiaoZhang; ZuLuYe; JianYong Shao

    2015-01-01

    Introduction:The rearrangement of the anaplastic lymphoma kinase (ALK) gene accounts for approximately 1%–6%of lung adenocarcinoma cases and deifnes a molecular subgroup of tumors characterized by clinical sensitivity toALK inhibitors such as crizotinib. This study aimed to identify the relationship betweenALK rearrangement and the clinico‑pathologic characteristics of non‑small cell lung cancer (NSCLC) and to analyze the therapeutic responses of crizotinib and conventional chemotherapy toALK rearrangement in NSCLC patients. Methods:A total of 487 lung cancer patients who underwent testing forALK rearrangement in our department were included in this study.ALK rearrangement was examined by using lfuorescence insitu hybridization (FISH) assay. Results:Among the 487 patients, 44 (9.0%) were diagnosed withALK rearrangement by using FISH assay. In 123 patients with adenocarcinoma who were non‑smokers and of a young age (≤58years old), the frequency ofALK rearrangement was 20.3% (25/123). Short overall survival (OS) was associated with non‑adenocarcinoma tumor type (P=0.006), poorly differentiated tumors (P=0.001), advanced‑stage tumors (P<0.001), smoking history (P=0.008), and wild‑type epidermal growth factor receptor (EGFR) (P=0.008). Moreover, patients with poorly differentiated and advanced‑stage tumors had a shorter time to cancer progression compared with those with well differentiated (P=0.023) and early‑stage tumors (P=0.001), respectively. Conclusions:ALK‑rearranged NSCLC tends to occur in younger individuals who are either non‑smokers or light smokers with adenocarcinoma. Patients withALK rearrangement might beneift fromALK inhibitor therapy.

  12. Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.

    Science.gov (United States)

    Wang, Xiaohong; Tolba, Emad; Schröder, Heinz C; Neufurth, Meik; Feng, Qingling; Diehl-Seifert, Bärbel; Müller, Werner E G

    2014-01-01

    We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP • Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2 : CaO : P2O5 of 55 : 40 : 5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP • Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP • Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP • Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide

  13. Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.

    Directory of Open Access Journals (Sweden)

    Xiaohong Wang

    Full Text Available We investigated the effect of bioglass (bioactive glass on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP, administered as polyP • Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nanoparticles, with a size of 55 nm and a molar ratio of SiO2 : CaO : P2O5 of 55 : 40 : 5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP • Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP • Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP • Ca2+-complex and 50 µmoles/L biosilica from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing

  14. 3D Hierarchical Pt-Nitrogen-Doped-Graphene-Carbonized Commercially Available Sponge as a Superior Electrocatalyst for Low-Temperature Fuel Cells.

    Science.gov (United States)

    Zhao, Lei; Sui, Xu-Lei; Li, Jia-Long; Zhang, Jing-Jia; Zhang, Li-Mei; Wang, Zhen-Bo

    2016-06-29

    Three-dimensional hierarchical nitrogen-doped graphene (3D-NG) frameworks were successfully fabricated through a feasible solution dip-coating method with commercially available sponges as the initial backbone. A spongy template can help hinder the graphene plates restacking in the period of the annealing process. The Pt/3D-NG catalyst was synthesized employing a polyol reduction process. The resultant Pt/3D-NG exhibits 2.3 times higher activity for methanol electro-oxidation along with the improvement in stability as compared with Pt/G owing to their favorable features including large specific surface area, high pore volume, high N doping level, and the homogeneous dispersion of Pt nanoparticles. Besides, Pt/3D-NG also presents high oxygen reduction reaction (ORR) performance in acid media when compared with Pt/3D-G and Pt/G. This work raises a valid solution for the fabrication of 3D functional freestanding graphene-based composites for a variety of applications in fuel cell catalysis, energy storage, and conversion.

  15. Coordinated rearrangements of assimilatory and storage cell compartments in a nitrogen-starving symbiotic chlorophyte cultivated under high light.

    Science.gov (United States)

    Gorelova, Olga; Baulina, Olga; Solovchenko, Alexei; Selyakh, Irina; Chivkunova, Olga; Semenova, Larisa; Scherbakov, Pavel; Burakova, Olga; Lobakova, Elena

    2015-03-01

    A quantitative micromorphometric study of the cell compartment rearrangements was performed in a symbiotic chlorophyte Desmodesmus sp. 3Dp86E-1 grown on nitrogen (N) replete or N-free medium under 480 μmol PAR quanta m(-2) s(-1). The changes in the chloroplast, intraplastidial, and cytoplasmic inclusions induced by high light (HL) and N starvation were similar to those characteristic of free-living chlorophytes. The N-sufficient culture responded to HL by a transient swelling of the thylakoid lumen and a decline in photosynthetic efficiency followed by its recovery. In the N-starving cells, a more rapid expansion and thylakoid swelling occurred along with the irreversible decline in the photosynthetic efficiency. Differential induction of starch grains, oil bodies, and cell wall polysaccharides depending on the stress exposure and type was recorded. Tight relationships between the changes in the assimilatory and storage compartments in the stressed Desmodesmus sp. cells were revealed.

  16. Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems.

    Science.gov (United States)

    Brajša, Karmen; Vujasinović, Ines; Jelić, Dubravko; Trzun, Marija; Zlatar, Ivo; Karminski-Zamola, Grace; Hranjec, Marijana

    2016-12-01

    Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure-activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.

  17. Regulation of adipose-tissue-derived stromal cell orientation and motility in 2D- and 3D-cultures by direct-current electrical field.

    Science.gov (United States)

    Yang, Gang; Long, Haiyan; Ren, Xiaomei; Ma, Kunlong; Xiao, Zhenghua; Wang, Ying; Guo, Yingqiang

    2017-02-01

    Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane-protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose-tissue-derived stromal cells (ADSCs) in 2D-culture on plastic culture dishes and in 3D-culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L-lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological-strength EFs in a homemade EF-bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time-lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D-culture aligned vertically to EF vector and kept a good cell survival rate. In 3D-culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D-culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors.

  18. The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

    Directory of Open Access Journals (Sweden)

    S Stephan

    2011-09-01

    Full Text Available The adult human intervertebral disc (IVD is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the influence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture.Bovine nucleus pulposus (NP cells were seeded at a range of cell densities (1.25 x105-106 cells/mL and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10 % serum, or without glucose and/or 20 % serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture.Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated “ghost” cells was positively correlated to cell seeding density. Increasing serum levels from 10 % to 20 % was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells. These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or “ghost” cells in vitro suggests that environmental factors may influence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

  19. Novel MAPK-dependent and -independent tubulogenes identified via microarray analysis of 3D-cultured Madin-Darby canine kidney cells.

    Science.gov (United States)

    Chacon-Heszele, Maria F; Zuo, Xiaofeng; Hellman, Nathan E; McKenna, Sarah; Choi, Soo Young; Huang, Liwei; Tobias, John W; Park, Kwon Moo; Lipschutz, Joshua H

    2014-05-01

    Cystogenesis and tubulogenesis are basic building blocks for many epithelial organs, including the kidney. Most researchers have used two-dimensional (2D) cell culture to investigate signaling pathways downstream of hepatocyte growth factor (HGF). We hypothesize that three-dimensional (3D) collagen-grown Madin-Darby canine kidney (MDCK) cells, which form cysts and then tubulate in response to HGF, are a much more in vivo-like system for the identification of novel tubulogenes. With the use of a canine microarray containing over 20,000 genes, 2,417 genes were identified as potential tubulogenes that were differentially regulated, exclusively in 3D-grown MDCK cells. Among these, 840 were dependent on MAPK signaling. Importantly, this work shows that many putative tubulogenes, previously identified via microarray analysis of 2D cultures, including by us, do not change in 3D culture and vice versa. The use of a 3D-culture system allowed for the identification of novel MAPK-dependent and -independent genes that regulate early renal tubulogenesis in vitro, e.g., matrix metalloproteinase 1 (MMP1). Knockdown of MMP1 led to defects in cystogenesis and tubulogenesis in 3D-grown MDCK cells, most likely due to problems establishing normal polarity. We suggest that data obtained from 2D cultures, even those using MDCK cells treated with HGF, should not be automatically extrapolated to factors important for cystogenesis and tubulogenesis. Instead, 3D culture, which more closely replicates the biological environment and is therefore a more accurate model for identifying tubulogenes, is preferred. Results from the present analysis will be used to build a more accurate model of the signaling pathways that control cystogenesis and tubulogenesis.

  20. Bioengineered 3D brain tumor model to elucidate the effects of matrix stiffness on glioblastoma cell behavior using PEG-based hydrogels.

    Science.gov (United States)

    Wang, Christine; Tong, Xinming; Yang, Fan

    2014-07-01

    Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor with a median survival of 12-15 months, and the mechanisms underlying GBM tumor progression remain largely elusive. Given the importance of tumor niche signaling in driving GBM progression, there is a strong need to develop in vitro models to facilitate analysis of brain tumor cell-niche interactions in a physiologically relevant and controllable manner. Here we report the development of a bioengineered 3D brain tumor model to help elucidate the effects of matrix stiffness on GBM cell fate using poly(ethylene-glycol) (PEG)-based hydrogels with brain-mimicking biochemical and mechanical properties. We have chosen PEG given its bioinert nature and tunable physical property, and the resulting hydrogels allow tunable matrix stiffness without changing the biochemical contents. To facilitate cell proliferation and migration, CRGDS and a MMP-cleavable peptide were chemically incorporated. Hyaluronic acid (HA) was also incorporated to mimic the concentration in the brain extracellular matrix. Using U87 cells as a model GBM cell line, we demonstrate that such biomimetic hydrogels support U87 cell growth, spreading, and migration in 3D over the course of 3 weeks in culture. Gene expression analyses showed U87 cells actively deposited extracellular matrix and continued to upregulate matrix remodeling genes. To examine the effects of matrix stiffness on GBM cell fate in 3D, we encapsulated U87 cells in soft (1 kPa) or stiff (26 kPa) hydrogels, which respectively mimics the matrix stiffness of normal brain or GBM tumor tissues. Our results suggest that changes in matrix stiffness induce differential GBM cell proliferation, morphology, and migration modes in 3D. Increasing matrix stiffness led to delayed U87 cell proliferation inside hydrogels, but cells formed denser spheroids with extended cell protrusions. Cells cultured in stiff hydrogels also showed upregulation of HA synthase 1 and matrix

  1. IZDELAVA TISKALNIKA 3D

    OpenAIRE

    Brdnik, Lovro

    2015-01-01

    Diplomsko delo analizira trenutno stanje 3D tiskalnikov na trgu. Prikazan je razvoj in principi delovanja 3D tiskalnikov. Predstavljeni so tipi 3D tiskalnikov, njihove prednosti in slabosti. Podrobneje je predstavljena zgradba in delovanje koračnih motorjev. Opravljene so meritve koračnih motorjev. Opisana je programska oprema za rokovanje s 3D tiskalniki in komponente, ki jih potrebujemo za izdelavo. Diploma se oklepa vprašanja, ali je izdelava 3D tiskalnika bolj ekonomična kot pa naložba v ...

  2. Composite System of Graphene Oxide and Polypeptide Thermogel As an Injectable 3D Scaffold for Adipogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Patel, Madhumita; Moon, Hyo Jung; Ko, Du Young; Jeong, Byeongmoon

    2016-03-02

    As two-dimensional (2D) nanomaterials, graphene (G) and graphene oxide (GO) have evolved into new platforms for biomedical research as biosensors, imaging agents, and drug delivery carriers. In particular, the unique surface properties of GO can be an important tool in modulating cellular behavior and various biological sequences. Here, we report that a composite system of graphene oxide/polypeptide thermogel (GO/P), prepared by temperature-sensitive sol-to-gel transition of a GO-suspended poly(ethylene glycol)-poly(L-alanine) (PEG-PA) aqueous solution significantly enhances the expression of adipogenic biomarkers, including PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, and HSL, compared to both a pure hydrogel system and a composite system of G/P, graphene-incorporated hydrogel. We prove that insulin, an adipogenic differentiation factor, preferentially adhered to GO, is supplied to the incorporated stem cells in a sustained manner over the three-dimensional (3D) cell culture period. On the other hand, insulin is partially denatured in the presence of G and interferes with the adipogenic differentiation of the stem cells. The study suggests that a 2D/3D composite system is a promising platform as a 3D cell culture matrix, where the surface properties of 2D materials in modulating the fates of the stem cells are effectively transcribed in a 3D culture system.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Matrix density alters zyxin phosphorylation, which limits peripheral process formation and extension in endothelial cells invading 3D collagen matrices.

    Science.gov (United States)

    Abbey, Colette A; Bayless, Kayla J

    2014-09-01

    This study was designed to determine the optimal conditions required for known pro-angiogenic stimuli to elicit successful endothelial sprouting responses. We used an established, quantifiable model of endothelial cell (EC) sprout initiation where ECs were tested for invasion in low (1 mg/mL) and high density (5 mg/mL) 3D collagen matrices. Sphingosine 1-phosphate (S1P) alone, or S1P combined with stromal derived factor-1α (SDF) and phorbol ester (TPA), elicited robust sprouting responses. The ability of these factors to stimulate sprouting was more effective in higher density collagen matrices. S1P stimulation resulted in a significant increase in invasion distance, and with the exception of treatment groups containing phorbol ester, invasion distance was longer in 1mg/mL compared to 5mg/mL collagen matrices. Closer examination of cell morphology revealed that increasing matrix density and supplementing with SDF and TPA enhanced the formation of multicellular structures more closely resembling capillaries. TPA enhanced the frequency and size of lumen formation and correlated with a robust increase in phosphorylation of p42/p44 Erk kinase, while S1P and SDF did not. Also, a higher number of significantly longer extended processes formed in 5mg/mL compared to 1mg/mL collagen matrices. Because collagen matrices at higher density have been reported to be stiffer, we tested for changes in the mechanosensitive protein, zyxin. Interestingly, zyxin phosphorylation levels inversely correlated with matrix density, while levels of total zyxin did not change significantly. Immunofluorescence and localization studies revealed that total zyxin was distributed evenly throughout invading structures, while phosphorylated zyxin was slightly more intense in extended peripheral processes. Silencing zyxin expression increased extended process length and number of processes, while increasing zyxin levels decreased extended process length. Altogether these data indicate that ECs

  5. Assembly of cell-laden hydrogel fiber into non-liquefied and liquefied 3D spiral constructs by perfusion-based layer-by-layer technique.

    Science.gov (United States)

    Sher, Praveen; Oliveira, Sara M; Borges, João; Mano, João F

    2015-01-06

    In this work, three-dimensional (3D) self-sustaining, spiral-shaped constructs were produced through a combination of ionotropic gelation, to form cell-encapsulated alginate fibers, and a perfusion-based layer-by-layer (LbL) technique. Single fibers were assembled over cylindrical molds by reeling to form spiral shapes, both having different geometries and sizes. An uninterrupted nanometric multilayer coating produced by a perfusion-based LbL technique, using alginate and chitosan, generated stable 3D spiral-shaped macrostructures by gripping and affixing the threads together without using any crosslinking/binding agent. The chelation process altered the internal microenvironment of the 3D construct from the solid to the liquefied state while preserving the external geometry. L929 cell viability by MTS and dsDNA quantification favor liquefied 3D constructs more than non-liquefied ones. The proposed technique setup helps us to generate complex polyelectrolyte-based 3D constructs for tissue engineering applications and organ printing.

  6. Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D.

    Science.gov (United States)

    Faulkner-Jones, Alan; Fyfe, Catherine; Cornelissen, Dirk-Jan; Gardner, John; King, Jason; Courtney, Aidan; Shu, Wenmiao

    2015-10-21

    We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.

  7. Rearrangement of mouse immunoglobulin kappa deleting element recombining sequence promotes immune tolerance and lambda B cell production.

    Science.gov (United States)

    Vela, José Luis; Aït-Azzouzene, Djemel; Duong, Bao Hoa; Ota, Takayuki; Nemazee, David

    2008-02-01

    The recombining sequence (RS) of mouse and its human equivalent, the immunoglobulin (Ig) kappa deleting element (IGKDE), are sequences found at the 3' end of the Ig kappa locus (Igk) that rearrange to inactivate Igk in developing B cells. RS recombination correlates with Ig lambda (Iglambda) light (L) chain expression and likely plays a role in receptor editing by eliminating Igk genes encoding autoantibodies. A mouse strain was generated in which the recombination signal of RS was removed, blocking RS-mediated Igk inactivation. In RS mutant mice, receptor editing and self-tolerance were impaired, in some cases leading to autoantibody formation. Surprisingly, mutant mice also made fewer B cells expressing lambda chain, whereas lambda versus kappa isotype exclusion was only modestly affected. These results provide insight into the mechanism of L chain isotype exclusion and indicate that RS has a physiological role in promoting the formation of lambda L chain-expressing B cells.

  8. A Rapid and Efficient 2D/3D Nuclear Segmentation Method for Analysis of Early Mouse Embryo and Stem Cell Image Data

    Directory of Open Access Journals (Sweden)

    Xinghua Lou

    2014-03-01

    Full Text Available Segmentation is a fundamental problem that dominates the success of microscopic image analysis. In almost 25 years of cell detection software development, there is still no single piece of commercial software that works well in practice when applied to early mouse embryo or stem cell image data. To address this need, we developed MINS (modular interactive nuclear segmentation as a MATLAB/C++-based segmentation tool tailored for counting cells and fluorescent intensity measurements of 2D and 3D image data. Our aim was to develop a tool that is accurate and efficient yet straightforward and user friendly. The MINS pipeline comprises three major cascaded modules: detection, segmentation, and cell position classification. An extensive evaluation of MINS on both 2D and 3D images, and comparison to related tools, reveals improvements in segmentation accuracy and usability. Thus, its accuracy and ease of use will allow MINS to be implemented for routine single-cell-level image analyses.

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

    CERN Document Server

    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...

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

    Science.gov (United States)

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

    2015-11-01

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

  11. 3D and Education

    Science.gov (United States)

    Meulien Ohlmann, Odile

    2013-02-01

    Today the industry offers a chain of 3D products. Learning to "read" and to "create in 3D" becomes an issue of education of primary importance. 25 years professional experience in France, the United States and Germany, Odile Meulien set up a personal method of initiation to 3D creation that entails the spatial/temporal experience of the holographic visual. She will present some different tools and techniques used for this learning, their advantages and disadvantages, programs and issues of educational policies, constraints and expectations related to the development of new techniques for 3D imaging. Although the creation of display holograms is very much reduced compared to the creation of the 90ies, the holographic concept is spreading in all scientific, social, and artistic activities of