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Sample records for 3d cell growth

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

    SciTech Connect

    Chen, Chih-Hao; Kuo, Shyh Ming; Liu, Guei-Sheung; Chen, Wan-Nan U.; Chuang, Chin-Wen; Liu, Li-Feng

    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.

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

    PubMed

    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.

  3. A Tunable Scaffold of Microtubular Graphite for 3D Cell Growth

    PubMed Central

    2016-01-01

    Aerographite (AG) is a novel carbon-based material that exists as a self-supportive 3D network of interconnected hollow microtubules. It can be synthesized in a variety of architectures tailored by the growth conditions. This flexibility in creating structures presents interesting bioengineering possibilities such as the generation of an artificial extracellular matrix. Here we have explored the feasibility and potential of AG as a scaffold for 3D cell growth employing cyclic RGD (cRGD) peptides coupled to poly(ethylene glycol) (PEG) conjugated phospholipids for surface functionalization to promote specific adhesion of fibroblast cells. Successful growth and invasion of the bulk material was followed over a period of 4 days. PMID:27258400

  4. 3D cell culture systems modeling tumor growth determinants in cancer target discovery.

    PubMed

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

    2014-04-01

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

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

    SciTech Connect

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

    2013-01-01

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

  6. Real-time 3D imaging of microstructure growth in battery cells using indirect MRI

    PubMed Central

    Ilott, Andrew J.; Mohammadi, Mohaddese; Chang, Hee Jung; Grey, Clare P.; Jerschow, Alexej

    2016-01-01

    Lithium metal is a promising anode material for Li-ion batteries due to its high theoretical specific capacity and low potential. The growth of dendrites is a major barrier to the development of high capacity, rechargeable Li batteries with lithium metal anodes, and hence, significant efforts have been undertaken to develop new electrolytes and separator materials that can prevent this process or promote smooth deposits at the anode. Central to these goals, and to the task of understanding the conditions that initiate and propagate dendrite growth, is the development of analytical and nondestructive techniques that can be applied in situ to functioning batteries. MRI has recently been demonstrated to provide noninvasive imaging methodology that can detect and localize microstructure buildup. However, until now, monitoring dendrite growth by MRI has been limited to observing the relatively insensitive metal nucleus directly, thus restricting the temporal and spatial resolution and requiring special hardware and acquisition modes. Here, we present an alternative approach to detect a broad class of metallic dendrite growth via the dendrites’ indirect effects on the surrounding electrolyte, allowing for the application of fast 3D 1H MRI experiments with high resolution. We use these experiments to reconstruct 3D images of growing Li dendrites from MRI, revealing details about the growth rate and fractal behavior. Radiofrequency and static magnetic field calculations are used alongside the images to quantify the amount of the growing structures. PMID:27621444

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-19

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

  9. 2D protrusion but not motility predicts growth factor-induced cancer cell migration in 3D collagen.

    PubMed

    Meyer, Aaron S; Hughes-Alford, Shannon K; Kay, Jennifer E; Castillo, Amalchi; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A

    2012-06-11

    Growth factor-induced migration is a critical step in the dissemination and metastasis of solid tumors. Although differences in properties characterizing cell migration on two-dimensional (2D) substrata versus within three-dimensional (3D) matrices have been noted for particular growth factor stimuli, the 2D approach remains in more common use as an efficient surrogate, especially for high-throughput experiments. We therefore were motivated to investigate which migration properties measured in various 2D assays might be reflective of 3D migratory behavioral responses. We used human triple-negative breast cancer lines stimulated by a panel of receptor tyrosine kinase ligands relevant to mammary carcinoma progression. Whereas 2D migration properties did not correlate well with 3D behavior across multiple growth factors, we found that increased membrane protrusion elicited by growth factor stimulation did relate robustly to enhanced 3D migration properties of the MDA-MB-231 and MDA-MB-157 lines. Interestingly, we observed this to be a more reliable relationship than cognate receptor expression or activation levels across these and two additional mammary tumor lines.

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

    PubMed

    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. A 3D Culture System Enhances the Ability of Human Bone Marrow Stromal Cells to Support the Growth of Limbal Stem/Progenitor Cells

    PubMed Central

    González, Sheyla; Mei, Hua; Nakatsu, Martin N.; Baclagon, Elfren R.; Deng, Sophie X.

    2016-01-01

    The standard method of cultivating limbal epithelial progenitor/stem cells (LSCs) on a monolayer of mouse 3T3 feeder cells possesses the risk of cross-contamination in clinical applications. Human feeder cells have been used to eliminate this risk; however, efficiency from xenobiotic-free cultures on a monolayer appears to be lower than in the standard method using 3T3 cells. We investigated whether bone marrow stromal cells (BMSCs), also known as bone marrow-derived mesenchymal stem cells, could serve as feeder cells for the expansion of LSCs in the 3-dimensional (3D) system. Primary single human LSCs on a monolayer of 3T3s served as the control. Very poor growth was observed when single LSCs were cultured on BMSCs. When LSC clusters were cultured on a BMSC monolayer (CC-BM), 3D culture system (3D CC-BM) and fibrin 3D system (fibrin 3D CC-BM), the 3D CC-BM method supported a greater LSC expansion. The 3D CC-BM system produced a 2.5-fold higher cell growth rate than the control (p<0.05). The proportion of K14+ and p63αbright cells were comparable to those in the control (p>0.05), whereas the proportion of K12+ cells was lower (p<0.05). These results indicate that BMSCs can efficiently support the expansion of the LSC population in the 3D culture. PMID:26896856

  12. Control of Retinal Ganglion Cell Positioning and Neurite Growth: Combining 3D Printing with Radial Electrospun Scaffolds.

    PubMed

    Kador, Karl E; Grogan, Shawn P; Dorthé, Erik W; Venugopalan, Praseeda; Malek, Monisha F; Goldberg, Jeffrey L; D'lima, Darryl D

    2016-02-01

    Retinal ganglion cells (RGCs) are responsible for the transfer of signals from the retina to the brain. As part of the central nervous system, RGCs are unable to regenerate following injury, and implanted cells have limited capacity to orient and integrate in vivo. During development, secreted guidance molecules along with signals from extracellular matrix and the vasculature guide cell positioning, for example, around the fovea, and axon outgrowth; however, these changes are temporally regulated and are not the same in the adult. Here, we combine electrospun cell transplantation scaffolds capable of RGC neurite guidance with thermal inkjet 3D cell printing techniques capable of precise positioning of RGCs on the scaffold surface. Optimal printing parameters are developed for viability, electrophysiological function and, neurite pathfinding. Different media, commonly used to promote RGC survival and growth, were tested under varying conditions. When printed in growth media containing both brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), RGCs maintained survival and normal electrophysiological function, and displayed radial axon outgrowth when printed onto electrospun scaffolds. These results demonstrate that 3D printing technology may be combined with complex electrospun surfaces in the design of future retinal models or therapies.

  13. Control of Retinal Ganglion Cell Positioning and Neurite Growth: Combining 3D Printing with Radial Electrospun Scaffolds

    PubMed Central

    Kador, Karl E.; Grogan, Shawn P.; Dorthé, Erik W.; Venugopalan, Praseeda; Malek, Monisha F.

    2016-01-01

    Retinal ganglion cells (RGCs) are responsible for the transfer of signals from the retina to the brain. As part of the central nervous system, RGCs are unable to regenerate following injury, and implanted cells have limited capacity to orient and integrate in vivo. During development, secreted guidance molecules along with signals from extracellular matrix and the vasculature guide cell positioning, for example, around the fovea, and axon outgrowth; however, these changes are temporally regulated and are not the same in the adult. Here, we combine electrospun cell transplantation scaffolds capable of RGC neurite guidance with thermal inkjet 3D cell printing techniques capable of precise positioning of RGCs on the scaffold surface. Optimal printing parameters are developed for viability, electrophysiological function and, neurite pathfinding. Different media, commonly used to promote RGC survival and growth, were tested under varying conditions. When printed in growth media containing both brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), RGCs maintained survival and normal electrophysiological function, and displayed radial axon outgrowth when printed onto electrospun scaffolds. These results demonstrate that 3D printing technology may be combined with complex electrospun surfaces in the design of future retinal models or therapies. PMID:26729061

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

    PubMed

    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.

  15. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src

    PubMed Central

    Torres-Ayuso, Pedro; Daza-Martín, Manuel; Martín-Pérez, Jorge; Ávila-Flores, Antonia; Mérida, Isabel

    2014-01-01

    Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer. PMID:25339152

  16. 3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

    PubMed

    Malenovská, Hana

    2016-02-01

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

  17. Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.

    PubMed

    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

  18. Effect of Bioglass on Growth and Biomineralization of SaOS-2 Cells in Hydrogel after 3D Cell Bioprinting

    PubMed Central

    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 a

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

    NASA Astrophysics Data System (ADS)

    Shamloo, Amir; Amirifar, Leyla

    2016-01-01

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies.

  20. 3D fold growth in transpression

    NASA Astrophysics Data System (ADS)

    Frehner, Marcel

    2016-12-01

    Geological folds in transpression are inherently 3D structures; hence their growth and rotation behavior is studied using 3D numerical finite-element simulations. Upright single-layer buckle folds in Newtonian materials are considered, which grow from an initial point-like perturbation due to a combination of in-plane shortening and shearing (i.e., transpression). The resulting fold growth exhibits three components: (1) fold amplification (vertical), (2) fold elongation (parallel to fold axis), and (3) sequential fold growth (perpendicular to axial plane) of new anti- and synforms adjacent to the initial fold. Generally, the fold growth rates are smaller for shearing-dominated than for shortening-dominated transpression. In spite of the growth rate, the folding behavior is very similar for the different convergence angles. The two lateral directions always exhibit similar growth rates implying that the bulk fold structure occupies an increasing roughly circular area. Fold axes are always parallel to the major horizontal principal strain axis (λ→max, i.e., long axis of the horizontal finite strain ellipse), which is initially also parallel to the major horizontal instantaneous stretching axis (ISA→max). After initiation, the fold axes rotate together with λ→max. Sequential folds appearing later do not initiate parallel to ISA→max, but parallel to λ→max, i.e. parallel to the already existing folds, and also rotate with λ→max. Therefore, fold axes do not correspond to passive material lines and hinge migration takes place as a consequence. The fold axis orientation parallel to λ→max is independent of convergence angle and viscosity ratio. Therefore, a triangular relationship between convergence angle, amount of shortening, and fold axis orientation exists. If two of these values are known, the third can be determined. This relationship is applied to the Zagros fold-and-thrust-belt to estimate the degree of strain partitioning between the Simply

  1. A 3D Hybrid Model for Tissue Growth: The Interplay between Cell Population and Mass Transport Dynamics

    PubMed Central

    Cheng, Gang; Markenscoff, Pauline; Zygourakis, Kyriacos

    2009-01-01

    Abstract To provide theoretical guidance for the design and in vitro cultivation of bioartificial tissues, we have developed a multiscale computational model that can describe the complex interplay between cell population and mass transport dynamics that governs the growth of tissues in three-dimensional scaffolds. The model has three components: a transient partial differential equation for the simultaneous diffusion and consumption of a limiting nutrient; a cellular automaton describing cell migration, proliferation, and collision; and equations that quantify how the varying nutrient concentration modulates cell division and migration. The hybrid discrete-continuous model was parallelized and solved on a distributed-memory multicomputer to study how transport limitations affect tissue regeneration rates under conditions encountered in typical bioreactors. Simulation results show that the severity of transport limitations can be estimated by the magnitude of two dimensionless groups: the Thiele modulus and the Biot number. Key parameters including the initial seeding mode, cell migration speed, and the hydrodynamic conditions in the bioreactor are shown to affect not only the overall rate, but also the pattern of tissue growth. This study lays the groundwork for more comprehensive models that can handle mixed cell cultures, multiple nutrients and growth factors, and other cellular processes, such as cell death. PMID:19619455

  2. Epidermal growth factor promotes a mesenchymal over an amoeboid motility of MDA-MB-231 cells embedded within a 3D collagen matrix

    NASA Astrophysics Data System (ADS)

    Geum, Dongil T.; Kim, Beum Jun; Chang, Audrey E.; Hall, Matthew S.; Wu, Mingming

    2016-01-01

    The receptor of epidermal growth factor (EGFR) critically regulates tumor cell invasion and is a potent therapeutic target for treatment of many types of cancers, including carcinomas and glioblastomas. It is known that EGF regulates cell motility when tumor cells are embedded within a 3D biomatrix. However, roles of EGF in modulating tumor cell motility phenotype are largely unknown. In this article, we report that EGF promotes a mesenchymal over an amoeboid motility phenotype using a malignant breast tumor cell line, MDA-MB-231, embedded within a 3D collagen matrix. Amoeboid cells are rounded in shape, while mesenchymal cells are elongated, and their migrations are governed by a distinctly different set of biomolecules. Using single cell tracking analysis, we also show that EGF promotes cell dissemination through a significant increase in cell persistence along with a moderate increase of speed. The increase of persistence is correlated with the increase of the percentage of the mesenchymal cells within the population. Our work reveals a novel role of microenvironmental cue, EGF, in modulating heterogeneity and plasticity of tumor cell motility phenotype. In addition, it suggests a potential visual cue for diagnosing invasive states of breast cancer cells. This work can be easily extended beyond breast cancer cells.

  3. Quantifying modes of 3D cell migration

    PubMed Central

    Driscoll, Meghan K.; Danuser, Gaudenz

    2015-01-01

    Although it is widely appreciated that cells migrate in a variety of diverse environments in vivo, we are only now beginning to use experimental workflows that yield images with sufficient spatiotemporal resolution to study the molecular processes governing cell migration in 3D environments. Since cell migration is a dynamic process, it is usually studied via microscopy, but 3D movies of 3D processes are difficult to interpret by visual inspection. In this review, we discuss the technologies required to study the diversity of 3D cell migration modes with a focus on the visualization and computational analysis tools needed to study cell migration quantitatively at a level comparable to the analyses performed today on cells crawling on flat substrates. PMID:26603943

  4. Quantifying Modes of 3D Cell Migration.

    PubMed

    Driscoll, Meghan K; Danuser, Gaudenz

    2015-12-01

    Although it is widely appreciated that cells migrate in a variety of diverse environments in vivo, we are only now beginning to use experimental workflows that yield images with sufficient spatiotemporal resolution to study the molecular processes governing cell migration in 3D environments. Since cell migration is a dynamic process, it is usually studied via microscopy, but 3D movies of 3D processes are difficult to interpret by visual inspection. In this review, we discuss the technologies required to study the diversity of 3D cell migration modes with a focus on the visualization and computational analysis tools needed to study cell migration quantitatively at a level comparable to the analyses performed today on cells crawling on flat substrates.

  5. Bioconductive 3D nano-composite constructs with tunable elasticity to initiate stem cell growth and induce bone mineralization.

    PubMed

    Sagar, Nitin; Khanna, Kunal; Sardesai, Varda S; Singh, Atul K; Temgire, Mayur; Kalita, Mridula Phukan; Kadam, Sachin S; Soni, Vivek P; Bhartiya, Deepa; Bellare, Jayesh R

    2016-12-01

    Bioactive 3D composites play an important role in advanced biomaterial design to provide molecular coupling and improve integrity with the cellular environment of the native bone. In the present study, a hybrid lyophilized polymer composite blend of anionic charged sodium salt of carboxymethyl chitin and gelatin (CMChNa-GEL) reinforced with nano-rod agglomerated hydroxyapatite (nHA) has been developed with enhanced biocompatibility and tunable elasticity. The scaffolds have an open, uniform and interconnected porous structure with an average pore diameter of 157±30μm and 89.47+0.03% with four dimensional X-ray. The aspect ratio of ellipsoidal pores decrease from 4.4 to 1.2 with increase in gelatin concentration; and from 2.14 to 1.93 with decrease in gelling temperature. The samples were resilient with elastic stain at 1.2MPa of stress also decreased from 0.33 to 0.23 with increase in gelatin concentration. The crosslinker HMDI (hexamethylene diisocyanate) yielded more resilient samples at 1.2MPa in comparison to glutaraldehyde. Increased crosslinking time from 2 to 4h in continuous compression cycle show no improvement in maximum elastic stain of 1.2MPa stress. This surface elasticity of the scaffold enables the capacity of these materials for adherent self renewal and cultivation of the NTERA-2 cL.D1 (NT2/D1), pluripotent embryonal carcinoma cell with biomechanical surface, as is shown here. Proliferation with MG-63, ALP activity and Alizarin red mineralization assay on optimized scaffold demonstrated ***p<0.001 between different time points thus showing its potential for bone healing. In pre-clinical study histological bone response of the scaffold construct displayed improved activity of bone regeneration in comparison to self healing of control groups (sham) up to week 07 after implantation in rabbit tibia critical-size defect. Therefore, this nHA-CMChNa-GEL scaffold composite exhibits inherent and efficient physicochemical, mechanical and biological

  6. 3D non-woven polyvinylidene fluoride scaffolds: fibre cross section and texturizing patterns have impact on growth of mesenchymal stromal cells.

    PubMed

    Schellenberg, Anne; Ross, Robin; Abagnale, Giulio; Joussen, Sylvia; Schuster, Philipp; Arshi, Annahit; Pallua, Norbert; Jockenhoevel, Stefan; Gries, Thomas; Wagner, Wolfgang

    2014-01-01

    Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds.

  7. Multizone Paper Platform for 3D Cell Cultures

    PubMed Central

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

    2011-01-01

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

  8. 3D Cell Culture in Alginate Hydrogels

    PubMed Central

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

    2015-01-01

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

  9. 3D culture for cardiac cells.

    PubMed

    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.

  10. Simulation of single-species bacterial-biofilm growth using the Glazier-Graner-Hogeweg model and the CompuCell3D modeling environment.

    PubMed

    Popławski, Nikodem J; Shirinifard, Abbas; Swat, Maciej; Glazier, James A

    2008-04-01

    The CompuCell3D modeling environment provides a convenient platform for biofilm simulations using the Glazier-Graner-Hogeweg (GGH) model, a cell-oriented framework designed to simulate growth and pattern formation due to biological cells' behaviors. We show how to develop such a simulation, based on the hybrid (continuum-discrete) model of Picioreanu, van Loosdrecht, and Heijnen (PLH), simulate the growth of a single-species bacterial biofilm, and study the roles of cell-cell and cell-field interactions in determining biofilm morphology. In our simulations, which generalize the PLH model by treating cells as spatially extended, deformable bodies, differential adhesion between cells, and their competition for a substrate (nutrient), suffice to produce a fingering instability that generates the finger shapes of biofilms. Our results agree with most features of the PLH model, although our inclusion of cell adhesion, which is difficult to implement using other modeling approaches, results in slightly different patterns. Our simulations thus provide the groundwork for simulations of medically and industrially important multispecies biofilms.

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

    PubMed

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

    2016-03-15

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

  12. Adhesion and growth of human bone marrow mesenchymal stem cells on precise-geometry 3D organic-inorganic composite scaffolds for bone repair.

    PubMed

    Chatzinikolaidou, Maria; Rekstyte, Sima; Danilevicius, Paulius; Pontikoglou, Charalampos; Papadaki, Helen; Farsari, Maria; Vamvakaki, Maria

    2015-03-01

    Engineering biomaterial scaffolds that promote attachment and growth of mesenchymal stem cells in three dimensions is a crucial parameter for successful bone tissue engineering. Towards this direction, a lot of research effort has focused recently into the development of three-dimensional porous scaffolds, aiming to elicit positive cellular behavior. However, the fabrication of three-dimensional tissue scaffolds with a precise geometry and complex micro- and nano-features, supporting cell in-growth remains a challenge. In this study we report on a positive cellular response of human bone marrow-derived (BM) mesenchymal stem cells (MSCs) onto hybrid material scaffolds consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide, and 2-(dimethylamino)ethyl methacrylate (DMAEMA). First, we use Direct fs Laser Writing, a 3D scaffolding technology to fabricate the complex structures. Subsequently, we investigate the morphology, viability and proliferation of BM-MSCs onto the hybrid scaffolds and examine the cellular response from different donors. Finally, we explore the effect of the materials' chemical composition on cell proliferation, employing three different material surfaces: (i) a hybrid consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide and 50mol% DMAEMA, (ii) a hybrid material comprising methacryloxypropyl trimethoxysilane and zirconium propoxide, and (iii) a purely organic polyDMAEMA. Our results show a strong adhesion of BM-MSCs onto the hybrid material containing 50% DMAEMA from the first 2h after seeding, and up to several days, and a proliferation increase after 14 and 21days, similar to the polystyrene control, independent of cell donor. These findings support the potential use of our proposed cell-material combination in bone tissue engineering.

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

    PubMed

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

    2016-07-01

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

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

    PubMed

    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.

  15. 3-D Volumetric Evaluation of Human Mandibular Growth

    PubMed Central

    Reynolds, Mathew; Reynolds, Michael; Adeeb, Samer; El-Bialy, Tarek

    2011-01-01

    Bone growth is a complex process that is controlled by a multitude of mechanisms that are not fully understood.Most of the current methods employed to measure the growth of bones focus on either studying cadaveric bones from different individuals of different ages, or successive two-dimensional (2D) radiographs. Both techniques have their known limitations. The purpose of this study was to explore a technique for quantifying the three dimensional (3D) growth of an adolescent human mandible over the period of one year utilizing cone beam computed tomography (CBCT) scans taken for regular orthodontic records. Three -dimensional virtual models were created from the CBCT data using mainstream medical imaging software. A comparison between computer-generated surface meshes of successive 3-D virtual models illustrates the magnitude of relative mandible growth. The results of this work are in agreement with previously reported data from human cadaveric studies and implantable marker studies. The presented method provides a new relatively simple basis (utilizing commercially available software) to visualize and evaluate individualized 3D (mandibular) growth in vivo. PMID:22046201

  16. 3-d volumetric evaluation of human mandibular growth.

    PubMed

    Reynolds, Mathew; Reynolds, Michael; Adeeb, Samer; El-Bialy, Tarek

    2011-01-01

    Bone growth is a complex process that is controlled by a multitude of mechanisms that are not fully understood.Most of the current methods employed to measure the growth of bones focus on either studying cadaveric bones from different individuals of different ages, or successive two-dimensional (2D) radiographs. Both techniques have their known limitations. The purpose of this study was to explore a technique for quantifying the three dimensional (3D) growth of an adolescent human mandible over the period of one year utilizing cone beam computed tomography (CBCT) scans taken for regular orthodontic records. Three -dimensional virtual models were created from the CBCT data using mainstream medical imaging software. A comparison between computer-generated surface meshes of successive 3-D virtual models illustrates the magnitude of relative mandible growth. The results of this work are in agreement with previously reported data from human cadaveric studies and implantable marker studies. The presented method provides a new relatively simple basis (utilizing commercially available software) to visualize and evaluate individualized 3D (mandibular) growth in vivo.

  17. 3D fold growth rates in transpressional tectonic settings

    NASA Astrophysics Data System (ADS)

    Frehner, Marcel

    2015-04-01

    Geological folds are inherently three-dimensional (3D) structures; hence, they also grow in 3D. In this study, fold growth in all three dimensions is quantified numerically using a finite-element algorithm for simulating deformation of Newtonian media in 3D. The presented study is an extension and generalization of the work presented in Frehner (2014), which only considered unidirectional layer-parallel compression. In contrast, the full range from strike slip settings (i.e., simple shear) to unidirectional layer-parallel compression is considered here by varying the convergence angle of the boundary conditions; hence the results are applicable to general transpressional tectonic settings. Only upright symmetrical single-layer fold structures are considered. The horizontal higher-viscous layer exhibits an initial point-like perturbation. Due to the mixed pure- and simple shear boundary conditions a mechanical buckling instability grows from this perturbation in all three dimensions, described by: Fold amplification (vertical growth): Fold amplification describes the growth from a fold shape with low limb-dip angle to a shape with higher limb-dip angle. Fold elongation (growth parallel to fold axis): Fold elongation describes the growth from a dome-shaped (3D) structure to a more cylindrical fold (2D). Sequential fold growth (growth perpendicular to fold axial plane): Sequential fold growth describes the growth of secondary (and further) folds adjacent to the initial isolated fold. The term 'lateral fold growth' is used as an umbrella term for both fold elongation and sequential fold growth. In addition, the orientation of the fold axis is tracked as a function of the convergence angle. Even though the absolute values of all three growth rates are markedly reduced with increasing simple-shear component at the boundaries, the general pattern of the quantified fold growth under the studied general-shear boundary conditions is surprisingly similar to the end

  18. Stem cell reprogramming: A 3D boost

    NASA Astrophysics Data System (ADS)

    Abilez, Oscar J.; Wu, Joseph C.

    2016-03-01

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

  19. Unit cell geometry of 3-D braided structures

    NASA Technical Reports Server (NTRS)

    Du, Guang-Wu; Ko, Frank K.

    1993-01-01

    The traditional approach used in modeling of composites reinforced by three-dimensional (3-D) braids is to assume a simple unit cell geometry of a 3-D braided structure with known fiber volume fraction and orientation. In this article, we first examine 3-D braiding methods in the light of braid structures, followed by the development of geometric models for 3-D braids using a unit cell approach. The unit cell geometry of 3-D braids is identified and the relationship of structural parameters such as yarn orientation angle and fiber volume fraction with the key processing parameters established. The limiting geometry has been computed by establishing the point at which yarns jam against each other. Using this factor makes it possible to identify the complete range of allowable geometric arrangements for 3-D braided preforms. This identified unit cell geometry can be translated to mechanical models which relate the geometrical properties of fabric preforms to the mechanical responses of composite systems.

  20. Towards Single Cell Traction Microscopy within 3D Collagen Matrices

    PubMed Central

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cells migration within collagen gels. PMID:23806281

  1. 3D Surface Topology Guides Stem Cell Adhesion and Differentiation

    PubMed Central

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

    2015-01-01

    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 stabilisers. 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. PMID:25818420

  2. Computational modeling of adherent cell growth in a hollow-fiber membrane bioreactor for large-scale 3-D bone tissue engineering.

    PubMed

    Mohebbi-Kalhori, Davod; Behzadmehr, Amin; Doillon, Charles J; Hadjizadeh, Afra

    2012-09-01

    The use of hollow-fiber membrane bioreactors (HFMBs) has been proposed for three-dimensional bone tissue growth at the clinical scale. However, to achieve an efficient HFMB design, the relationship between cell growth and environmental conditions must be determined. Therefore, in this work, a dynamic double-porous media model was developed to determine nutrient-dependent cell growth for bone tissue formation in a HFMB. The whole hollow-fiber scaffold within the bioreactor was treated as a porous domain in this model. The domain consisted of two interpenetrating porous regions, including a porous lumen region available for fluid flow and a porous extracapillary space filled with a collagen gel that contained adherent cells for promoting long-term growth into tissue-like mass. The governing equations were solved numerically and the model was validated using previously published experimental results. The contributions of several bioreactor design and process parameters to the performance of the bioreactor were studied. The results demonstrated that the process and design parameters of the HFMB significantly affect nutrient transport and thus cell behavior over a long period of culture. The approach presented here can be applied to any cell type and used to develop tissue engineering hollow-fiber scaffolds.

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

    PubMed Central

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

    2013-01-01

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

  4. A novel mechanotactic 3D modeling of cell morphology

    NASA Astrophysics Data System (ADS)

    Jamaleddin Mousavi, Seyed; Hamdy Doweidar, Mohamed

    2014-08-01

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

  5. "Constructing" the Cell Cycle in 3D

    ERIC Educational Resources Information Center

    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…

  6. Myosin IIA dependent retrograde flow drives 3D cell migration.

    PubMed

    Shih, Wenting; Yamada, Soichiro

    2010-04-21

    Epithelial cell migration is an essential part of embryogenesis and tissue regeneration, yet their migration is least understood. Using our three-dimensional (3D) motility analysis, migrating epithelial cells formed an atypical polarized cell shape with the nucleus leading the cell front and a contractile cell rear. Migrating epithelial cells exerted traction forces to deform both the anterior and posterior extracellular matrix toward the cell body. The cell leading edge exhibited a myosin II-dependent retrograde flow with the magnitude and direction consistent with surrounding network deformation. Interestingly, on a two-dimensional substrate, myosin IIA-deficient cells migrated faster than wild-type cells, but in a 3D gel, these myosin IIA-deficient cells were unpolarized and immobile. In contrast, the migration rates of myosin IIB-deficient cells were similar to wild-type cells. Therefore, myosin IIA, not myosin IIB, is required for 3D epithelial cell migration.

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

    PubMed

    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.

  8. Modeling cell migration in 3D: Status and challenges.

    PubMed

    Rangarajan, Rajagopal; Zaman, Muhammad H

    2008-01-01

    Cell migration is a multi-scale process that integrates signaling, mechanics and biochemical reaction kinetics. Various mathematical models accurately predict cell migration on 2D surfaces, but are unable to capture the complexities of 3D migration. Additionally, quantitative 3D cell migration models have been few and far between. In this review we look and characterize various mathematical models available in literature to predict cell migration in 3D matrices and analyze their strengths and possible changes to these models that could improve their predictive capabilities.

  9. Toward single cell traction microscopy within 3D collagen matrices

    SciTech Connect

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  10. Digital holographic microscopy for imaging growth and treatment response in 3D tumor models

    NASA Astrophysics Data System (ADS)

    Li, Yuyu; Petrovic, Ljubica; Celli, Jonathan P.; Yelleswarapu, Chandra S.

    2014-03-01

    While three-dimensional tumor models have emerged as valuable tools in cancer research, the ability to longitudinally visualize the 3D tumor architecture restored by these systems is limited with microscopy techniques that provide only qualitative insight into sample depth, or which require terminal fixation for depth-resolved 3D imaging. Here we report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, non-destructive longitudinal imaging of in vitro 3D tumor models. Following established methods we prepared 3D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple timepoints throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify nodule thickness over time under normal growth, and in cultures subject to chemotherapy treatment. In this manner total nodule volumes are rapidly estimated and demonstrated here to show contrasting time dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3D structure over time and suggests the further development of this approach for time-lapse monitoring of 3D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.

  11. Surface modified alginate microcapsules for 3D cell culture

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  12. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    PubMed

    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.

  13. An Open Source Image Processing Method to Quantitatively Assess Tissue Growth after Non-Invasive Magnetic Resonance Imaging in Human Bone Marrow Stromal Cell Seeded 3D Polymeric Scaffolds

    PubMed Central

    Leferink, Anne M.; Fratila, Raluca M.; Koenrades, Maaike A.; van Blitterswijk, Clemens A.; Velders, Aldrik; Moroni, Lorenzo

    2014-01-01

    Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional (3D) scaffolds for regenerative medicine and clinical purposes. This is even more important when multipotent human bone marrow stromal cells (hMSCs) are used, as it could offer a method to understand in real time the dynamics of stromal cell differentiation and eventually steer it into the desired lineage. Magnetic Resonance Imaging (MRI) is a promising tool to overcome the challenge of a limited transparency in opaque 3D scaffolds. Technical limitations of MRI involve non-uniform background intensity leading to fluctuating background signals and therewith complicating quantifications on the retrieved images. We present a post-imaging processing sequence that is able to correct for this non-uniform background intensity. To test the processing sequence we investigated the use of MRI for in vitro monitoring of tissue growth in three-dimensional poly(ethylene oxide terephthalate)–poly(butylene terephthalate) (PEOT/PBT) scaffolds. Results showed that MRI, without the need to use contrast agents, is a promising non-invasive tool to quantitatively monitor ECM production and cell distribution during in vitro culture in 3D porous tissue engineered constructs. PMID:25502022

  14. An open source image processing method to quantitatively assess tissue growth after non-invasive magnetic resonance imaging in human bone marrow stromal cell seeded 3D polymeric scaffolds.

    PubMed

    Leferink, Anne M; Fratila, Raluca M; Koenrades, Maaike A; van Blitterswijk, Clemens A; Velders, Aldrik; Moroni, Lorenzo

    2014-01-01

    Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional (3D) scaffolds for regenerative medicine and clinical purposes. This is even more important when multipotent human bone marrow stromal cells (hMSCs) are used, as it could offer a method to understand in real time the dynamics of stromal cell differentiation and eventually steer it into the desired lineage. Magnetic Resonance Imaging (MRI) is a promising tool to overcome the challenge of a limited transparency in opaque 3D scaffolds. Technical limitations of MRI involve non-uniform background intensity leading to fluctuating background signals and therewith complicating quantifications on the retrieved images. We present a post-imaging processing sequence that is able to correct for this non-uniform background intensity. To test the processing sequence we investigated the use of MRI for in vitro monitoring of tissue growth in three-dimensional poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) scaffolds. Results showed that MRI, without the need to use contrast agents, is a promising non-invasive tool to quantitatively monitor ECM production and cell distribution during in vitro culture in 3D porous tissue engineered constructs.

  15. 3D printing of biomimetic microstructures for cancer cell migration.

    PubMed

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

    2014-02-01

    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. 10 T1/2). 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 10 T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10 T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10 T1/2 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.

  16. 3D printing of biomimetic microstructures for cancer cell migration

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Cell proliferation and migration in silk fibroin 3D scaffolds.

    PubMed

    Mandal, Biman B; Kundu, Subhas C

    2009-05-01

    Pore architecture in 3D polymeric scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for the seeded cells to organize into a functioning tissue. In this report, we investigated the effects of different freezing temperature regimes on silk fibroin protein 3D scaffold pore microstructure. The fabricated scaffolds using freeze-dry technique were used as a 3D model to monitor cell proliferation and migration. Pores of 200-250microm diameter were formed by slow cooling at temperatures of -20 and -80 degrees C but were found to be limited in porosity and pore interconnectivity as observed through scanning electron microscopic images. In contrast, highly interconnected pores with 96% porosity were observed when silk solutions were rapidly frozen at -196 degrees C. A detailed study was conducted to assess the affect of pore size, porosity and interconnectivity on human dermal fibroblast cell proliferation and migration on these 3D scaffolds using confocal microscopy. The cells were observed to migrate within the scaffold interconnectivities and were found to reach scaffold periphery within 28 days of culture. Confocal images further confirmed normal cell attachment and alignment of actin filaments within the porous scaffold matrix with well-developed nuclei. This study indicates rapid freeze-drying technique as an alternative method to fabricate highly interconnected porous scaffolds for developing functional 3D silk fibroin matrices for potential tissue engineering, biomedical and biotechnological applications.

  19. 3D Protein Dynamics in the Cell Nucleus.

    PubMed

    Singh, Anand P; Galland, Rémi; Finch-Edmondson, Megan L; Grenci, Gianluca; Sibarita, Jean-Baptiste; Studer, Vincent; Viasnoff, Virgile; Saunders, Timothy E

    2017-01-10

    The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.

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

    SciTech Connect

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

    2010-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  2. 3D map of the human corneal endothelial cell

    PubMed Central

    He, Zhiguo; Forest, Fabien; Gain, Philippe; Rageade, Damien; Bernard, Aurélien; Acquart, Sophie; Peoc’h, Michel; Defoe, Dennis M.; Thuret, Gilles

    2016-01-01

    Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions. PMID:27381832

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

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Montanez-Sauri, Sara I.

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

  5. Dual-stage growth factor release within 3D protein-engineered hydrogel niches promotes adipogenesis

    PubMed Central

    Greenwood-Goodwin, Midori; Teasley, Eric S.; Heilshorn, Sarah C.

    2014-01-01

    Engineered biomimetic microenvironments from hydrogels are an emerging strategy to achieve lineage-specific differentiation in vitro. In addition to recapitulating critical matrix cues found in the native three-dimensional (3D) niche, the hydrogel can also be designed to deliver soluble factors that are present within the native inductive microenvironment. We demonstrate a versatile materials approach for the dual-stage delivery of multiple soluble factors within a 3D hydrogel to induce adipogenesis. We use a Mixing-Induced Two-Component Hydrogel (MITCH) embedded with alginate microgels to deliver two pro-adipogenic soluble factors, fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) with two distinct delivery profiles. We show that dual-stage delivery of FGF-1 and BMP-4 to human adipose-derived stromal cells (hADSCs) significantly increases lipid accumulation compared with the simultaneous delivery of both growth factors together. Furthermore, dual-stage growth factor delivery within a 3D hydrogel resulted in substantially more lipid accumulation compared to identical delivery profiles in 2D cultures. Gene expression analysis shows upregulation of key adipogenic markers indicative of brown-like adipocytes. These data suggest that dual-stage release of FGF-1 and BMP-4 within 3D microenvironments can promote the in vitro development of mature adipocytes. PMID:25309741

  6. Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models.

    PubMed

    Yue, Xiaoshan; Lukowski, Jessica K; Weaver, Eric M; Skube, Susan B; Hummon, Amanda B

    2016-12-02

    Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.

  7. Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays.

    PubMed

    Lal-Nag, Madhu; McGee, Lauren; Titus, Steven A; Brimacombe, Kyle; Michael, Sam; Sittampalam, Gurusingham; Ferrer, Marc

    2017-03-01

    Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.

  8. 3D Reconstruction of Coronary Artery Vascular Smooth Muscle Cells

    PubMed Central

    Luo, Tong; Chen, Huan; Kassab, Ghassan S.

    2016-01-01

    Aims 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. Methods and Results 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±14.9μm, 4.6±0.6μm and 6.2±1.8μm (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°. Conclusions 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. PMID:26882342

  9. Grid cells in 3-D: Reconciling data and models.

    PubMed

    Horiuchi, Timothy K; Moss, Cynthia F

    2015-12-01

    It is well documented that place cells and grid cells in echolocating bats show properties similar to those described in rodents, and yet, continuous theta-frequency oscillations, proposed to play a central role in grid/place cell formation, are not present in bat recordings. These comparative neurophysiological data have raised many questions about the role of theta-frequency oscillations in spatial memory and navigation. Additionally, spatial navigation in three-dimensions poses new challenges for the representation of space in neural models. Inspired by the literature on space representation in the echolocating bat, we have developed a nonoscillatory model of 3-D grid cell creation that shares many of the features of existing oscillatory-interference models. We discuss the model in the context of current knowledge of 3-D space representation and highlight directions for future research.

  10. Microfluidic titer plate for stratified 3D cell culture.

    PubMed

    Trietsch, Sebastiaan J; Israëls, Guido D; Joore, Jos; Hankemeier, Thomas; Vulto, Paul

    2013-09-21

    Human tissues and organs are inherently heterogeneous. Their functionality is determined by the interplay between different cell types, their secondary architecture, vascular system and gradients of signaling molecules and metabolites. Here we propose a stratified 3D cell culture platform, in which adjacent lanes of gels and liquids are patterned by phaseguides to capture this tissue heterogeneity. We demonstrate 3D cell culture of HepG2 hepatocytes under continuous perfusion, a rifampicin toxicity assay and co-culture with fibroblasts. 4T1 breast cancer cells are used to demonstrate invasion and aggregation models. The platform is incorporated in a microtiter plate format that renders it fully compatible with automation and high-content screening equipment. The extended functionality, ease of handling and full compatibility to standard equipment is an important step towards adoption of Organ-on-a-Chip technology for screening in an industrial setting.

  11. Fabricating gradient hydrogel scaffolds for 3D cell culture.

    PubMed

    Chatterjee, Kaushik; Young, Marian F; Simon, Carl G

    2011-05-01

    Optimizing cell-material interactions is critical for maximizing regeneration in tissue engineering. Combinatorial and high-throughput (CHT) methods can be used to systematically screen tissue scaffolds to identify optimal biomaterial properties. Previous CHT platforms in tissue engineering have involved a two-dimensional (2D) cell culture format where cells were cultured on material surfaces. However, these platforms are inadequate to predict cellular response in a three-dimensional (3D) tissue scaffold. We have developed a simple CHT platform to screen cell-material interactions in 3D culture format that can be applied to screen hydrogel scaffolds. Herein we provide detailed instructions on a method to prepare gradients in elastic modulus of photopolymerizable hydrogels.

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

    PubMed

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

    2014-12-15

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

  13. Protrusive waves guide 3D cell migration along nanofibers

    PubMed Central

    Guetta-Terrier, Charlotte; Monzo, Pascale; Zhu, Jie; Long, Hongyan; Venkatraman, Lakshmi; Zhou, Yue; Wang, PeiPei; Chew, Sing Yian; Mogilner, Alexander

    2015-01-01

    In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions. PMID:26553933

  14. Protrusive waves guide 3D cell migration along nanofibers.

    PubMed

    Guetta-Terrier, Charlotte; Monzo, Pascale; Zhu, Jie; Long, Hongyan; Venkatraman, Lakshmi; Zhou, Yue; Wang, PeiPei; Chew, Sing Yian; Mogilner, Alexander; Ladoux, Benoit; Gauthier, Nils C

    2015-11-09

    In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions.

  15. Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling.

    PubMed

    Syed, Deeba N; Chamcheu, Jean-Christopher; Khan, Mohammad Imran; Sechi, Mario; Lall, Rahul K; Adhami, Vaqar M; Mukhtar, Hasan

    2014-06-01

    The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways.

  16. Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling

    PubMed Central

    Syed, Deeba N.; Chamcheu, Jean-Christopher; Khan, Mohammad Imran; Sechi, Mario; Lall, Rahul K.; Adhami, Vaqar M.; Mukhtar, Hasan

    2014-01-01

    The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways. PMID:24675012

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

    PubMed Central

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

    2016-01-01

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

  18. 3D visualization of membrane failures in fuel cells

    NASA Astrophysics Data System (ADS)

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

    PubMed

    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.

  20. 3D cancer cell migration in a confined matrix

    NASA Astrophysics Data System (ADS)

    Alobaidi, Amani; Sun, Bo

    Cancer cell migration is widely studied in 2D motion, which does not mimic the invasion processes in vivo. More recently, 3D cell migration studies have been performed. The ability of cancer cells to migrate within the extracellular matrix depends on the physical and biochemical features of the extracellular matrix. We present a model of cell motility in confined matrix geometry. The aim of the study is to study cancer migration in collagen matrix, as a soft tissue, to investigate their motility within the confined and surrounding collagen environment. Different collagen concentrations have been used to show the ability of these cancer cells to move through such a complex structure by measuring Cancer cell migration velocity as well as the displacement. Graduate student physics department.

  1. Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments

    PubMed Central

    Welf, Erik S.; Driscoll, Meghan K.; Dean, Kevin M.; Schäfer, Claudia; Chu, Jun; Davidson, Michael W.; Lin, Michael Z.; Danuser, Gaudenz; Fiolka, Reto

    2016-01-01

    The microenvironment determines cell behavior, but the underlying molecular mechanisms are poorly understood because quantitative studies of cell signaling and behavior have been challenging due to insufficient spatial and/or temporal resolution and limitations on microenvironmental control. Here we introduce microenvironmental selective plane illumination microscopy (meSPIM) for imaging and quantification of intracellular signaling and submicrometer cellular structures as well as large-scale cell morphological and environmental features. We demonstrate the utility of this approach by showing that the mechanical properties of the microenvironment regulate the transition of melanoma cells from actin-driven protrusion to blebbing, and we present tools to quantify how cells manipulate individual collagen fibers. We leverage the nearly isotropic resolution of meSPIM to quantify the local concentration of actin and phosphatidylinositol 3-kinase signaling on the surfaces of cells deep within 3D collagen matrices and track the many small membrane protrusions that appear in these more physiologically relevant environments. PMID:26906741

  2. A 3D-printed local drug delivery patch for pancreatic cancer growth suppression.

    PubMed

    Yi, Hee-Gyeong; Choi, Yeong-Jin; Kang, Kyung Shin; Hong, Jung Min; Pati, Ruby Gupta; Park, Moon Nyeo; Shim, In Kyong; Lee, Chan Mi; Kim, Song Cheol; Cho, Dong-Woo

    2016-09-28

    Since recurrence and metastasis of pancreatic cancer has a worse prognosis, chemotherapy has been typically performed to attack the remained malignant cells after resection. However, it is difficult to achieve the therapeutic concentration at the tumor site with systemic chemotherapy. Numerous local drug delivery systems have been studied to overcome the shortcomings of systemic delivery. However, because most systems involve dissolution of the drug within the carrier, the concentration of the drug is limited to the saturation solubility, and consequently cannot reach the sufficient drug dose. Therefore, we hypothesized that 3D printing of a biodegradable patch incorporated with a high drug concentration would provide a versatile shape to be administered at the exact tumor site as well as an appropriate therapeutic drug concentration with a controlled release. Here, we introduce the 3D-printed patches composed of a blend of poly(lactide-co-glycolide), polycaprolactone, and 5-fluorouracil for delivering the anti-cancer drug in a prolonged controlled manner and therapeutic dose. 3D printing technology can manipulate the geometry of the patch and the drug release kinetics. The patches were flexible, and released the drug over four weeks, and thereby suppressed growth of the subcutaneous pancreatic cancer xenografts in mice with minimized side effects. Our approach reveals that 3D printing of bioabsorbable implants containing anti-cancer drugs could be a powerful method for an effective local delivery of chemotherapeutic agents to treatment of cancers.

  3. Vinculin Regulates Directionality and Cell Polarity in 2D, 3D Matrix and 3D Microtrack Migration.

    PubMed

    Rahman, Aniqua; Carey, Shawn P; Kraning-Rush, Casey M; Goldblatt, Zachary E; Bordeleau, Francois; Lampi, Marsha C; Lin, Deanna Y; García, Andrés J; Reinhart-King, Cynthia A

    2016-03-09

    During metastasis, cells can use proteolytic activity to form tube-like "microtracks" within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro 3D micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Since focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on 2D substrates and in 3D uniform collagen matrices, indicated by reduced speed, shorter net displacement and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for Focal Adhesion Kinase (FAK) activation in 3D as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks, but not on 2D substrates, and accordingly, FAK inhibition halts cell migration in 3D microtracks. Together, these data indicate that vinculin plays a key role in polarization during migration.

  4. Large-scale pharmacological profiling of 3D tumor models of cancer cells

    PubMed Central

    Mathews Griner, Lesley A; Zhang, Xiaohu; Guha, Rajarshi; McKnight, Crystal; Goldlust, Ian S; Lal-Nag, Madhu; Wilson, Kelli; Michael, Sam; Titus, Steve; Shinn, Paul; Thomas, Craig J; Ferrer, Marc

    2016-01-01

    The discovery of chemotherapeutic agents for the treatment of cancer commonly uses cell proliferation assays in which cells grow as two-dimensional (2D) monolayers. Compounds identified using 2D monolayer assays often fail to advance during clinical development, most likely because these assays do not reproduce the cellular complexity of tumors and their microenvironment in vivo. The use of three-dimensional (3D) cellular systems have been explored as enabling more predictive in vitro tumor models for drug discovery. To date, small-scale screens have demonstrated that pharmacological responses tend to differ between 2D and 3D cancer cell growth models. However, the limited scope of screens using 3D models has not provided a clear delineation of the cellular pathways and processes that differentially regulate cell survival and death in the different in vitro tumor models. Here we sought to further understand the differences in pharmacological responses between cancer tumor cells grown in different conditions by profiling a large collection of 1912 chemotherapeutic agents. We compared pharmacological responses obtained from cells cultured in traditional 2D monolayer conditions with those responses obtained from cells forming spheres versus cells already in 3D spheres. The target annotation of the compound library screened enabled the identification of those key cellular pathways and processes that when modulated by drugs induced cell death in all growth conditions or selectively in the different cell growth models. In addition, we also show that many of the compounds targeting these key cellular functions can be combined to produce synergistic cytotoxic effects, which in many cases differ in the magnitude of their synergism depending on the cellular model and cell type. The results from this work provide a high-throughput screening framework to profile the responses of drugs both as single agents and in pairwise combinations in 3D sphere models of cancer cells. PMID

  5. Controlled architectural and chemotactic studies of 3D cell migration.

    PubMed

    Tayalia, Prakriti; Mazur, Eric; Mooney, David J

    2011-04-01

    Chemotaxis plays a critical role in tissue development and wound repair, and is widely studied using ex vivo model systems in applications such as immunotherapy. However, typical chemotactic models employ 2D systems that are less physiologically relevant or use end-point assays, that reveal little about the stepwise dynamics of the migration process. To overcome these limitations, we developed a new model system using microfabrication techniques, sustained drug delivery approaches, and theoretical modeling of chemotactic agent diffusion. This model system allows us to study the effects of 3D architecture and chemotactic agent gradient on immune cell migration in real time. We find that dendritic cell migration is characterized by a strong interplay between matrix architecture and chemotactic gradients, and migration is also influenced dramatically by the cell activation state. Our results indicate that Lipopolysaccharide-activated dendritic cells studied in a traditional transwell system actually exhibit anomalous migration behavior. Such a 3D ex vivo system lends itself for analyzing cell migratory behavior in response to single or multiple competitive cues and could prove useful in vaccine development.

  6. 3D-printed external light trap for solar cells.

    PubMed

    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.

  7. Collective Motion of Mammalian Cell Cohorts in 3D

    PubMed Central

    Sharma, Yasha; Vargas, Diego A.; Pegoraro, Adrian F.; Lepzelter, David; Weitz, David A.; Zaman, Muhammad H

    2016-01-01

    Collective cell migration is ubiquitous in biology, from development to cancer; it occurs in complex systems comprised of heterogeneous cell types, signals and matrices, and requires large scale regulation in space and time. Understanding how cells achieve organized collective motility is crucial to addressing cellular and tissue function and disease progression. While current two-dimensional model systems recapitulate the dynamic properties of collective cell migration, quantitative three-dimensional equivalent model systems have proved elusive. To establish such a model system, we study cell collectives by tracking individuals within cell cohorts embedded in three dimensional collagen scaffolding. We develop a custom algorithm to quantify the temporal and spatial heterogeneity of motion in cell cohorts during motility events. In the absence of external driving agents, we show that these cohorts rotate in short bursts, <2 hours, and translate for up to 6 hours. We observe, track, and analyze three dimensional motion of cell cohorts composed of 3–31 cells, and pave a path toward understanding cell collectives in 3D as a complex emergent system. PMID:26549557

  8. Design of 3D engineered protein hydrogels for tailored control of neurite growth

    PubMed Central

    Lampe, Kyle J.; Antaris, Alexander L.; Heilshorn, Sarah C.

    2013-01-01

    The design of bioactive materials allows for tailored studies probing cell-biomaterial interactions; however, relatively few studies have examined effects of ligand density and material stiffness on neurite growth in 3D. Elastin-like proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within 3D materials. To promote neurite outgrowth and better understand the effects of common biomaterial design parameters on neuronal cultures, we here focused on cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of engineered proteins, these 3D ELP hydrogels enabled decoupled investigation into the effects of biomechanics and biochemistry on neurite outgrowth from dorsal root ganglia (DRG). Increasing the cell-adhesive RGD ligand density from 0 to 1.9 × 107 ligands/μm3 led to a significant increase in the rate, length, and density of neurite outgrowth, as quantified by a high-throughput algorithm developed for dense neurite analysis. An approximately two-fold improvement in total neurite outgrowth was observed in materials with the higher ligand density at all time-points through 7 days. ELP hydrogels with initial elastic moduli of 0.5, 1.5, or 2.1 kPa and identical RGD ligand densities revealed that the most compliant materials led to the greatest outgrowth, with some neurites extending over 1800 μm by day 7. Given the ability of ELP hydrogels to efficiently promote neurite outgrowth within defined and tunable 3D microenvironments, these materials may be useful in developing therapeutic nerve guides and the further study of basic neuron-biomaterial interactions. PMID:23128159

  9. 3D motion analysis of keratin filaments in living cells

    NASA Astrophysics Data System (ADS)

    Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf; Aach, Til

    2010-03-01

    We present a novel and efficient approach for 3D motion estimation of keratin intermediate filaments in vitro. Keratin filaments are elastic cables forming a complex scaffolding within epithelial cells. To understand the mechanisms of filament formation and network organisation under physiological and pathological conditions, quantitative measurements of dynamic network alterations are essential. Therefore we acquired time-lapse series of 3D images using a confocal laser scanning microscope. Based on these image series, we show that a dense vector field can be computed such that the displacements from one frame to the next can be determined. Our method is based on a two-step registration process: First, a rigid pre-registration is applied in order to compensate for possible global cell movement. This step enables the subsequent nonrigid registration to capture only the sought local deformations of the filaments. As the transformation model of the deformable registration algorithm is based on Free Form Deformations, it is well suited for modeling filament network dynamics. The optimization is performed using efficient linear programming techniques such that the huge amount of image data of a time series can be efficiently processed. The evaluation of our results illustrates the potential of our approach.

  10. XBP1-LOX Axis is critical in ER stress-induced growth of lung adenocarcinoma in 3D culture

    PubMed Central

    Yang, Yi; Cheng, Bai-Jun; Jian, Hong; Chen, Zhi-Wei; Zhao, Yi; Yu, Yong-Feng; Li, Zi-Ming; Liao, Mei-Lin; Lu, Shun

    2017-01-01

    Rapid growth of tumor cells needs to consume large amounts of oxygen and glucose, due to lack of blood supply within the tumor, cells live in an environment that lack of oxygen and nutrients. This environment results in endoplasmic reticulum (ER) stress and activates the UPR (unfolded protein response). More and more evidence suggests UPR provides a growth signal pathway required for tumor growth. In the present study, we investigated the relationship between XBP1, one transcription factor in UPR, and the expression of LOX. We found that ER stress induces high expression of XBP1, one transcription factor in UPR, in both 2D culture and 3D culture; but only promotes growth of lung adenocarcinoma cells in in vitro 3D culture other than 2D culture. In 3D culture, we further showed that knockdown XBP1 expression can block Tm/Tg-induced cell growth. LOX genes may be key downstream effector of XBP1. Knockdown LOX expression can partially block XBP1-induced cell growth. Then we showed XBP1 suppressed by RNA interference (RNAi) can reduce the expression of LOX. For the first time, it is being shown that XBP1 can regulate the expression of LOX to promote cell growth.

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

    PubMed

    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.

  12. Quasi-horizontal circulation cells in 3D seawater intrusion

    USGS Publications Warehouse

    Abarca, E.; Carrera, J.; Sanchez-Vila, X.; Voss, C.I.

    2007-01-01

    The seawater intrusion process is characterized by the difference in freshwater and seawater density that causes freshwater to float on seawater. Many confined aquifers have a large horizontal extension with respect to thickness. In these cases, while buoyancy acts in the vertical direction, flow is confined between the upper and bottom boundaries and the effect of gravity is controlled by variations of aquifer elevation. Therefore, the effective gravity is controlled by the slope and the shape of the aquifer boundaries. Variability in the topography of the aquifer boundaries is one case where 3D analysis is necessary. In this work, density-dependent flow processes caused by 3D aquifer geometry are studied numerically and specifically, considering a lateral slope of the aquifer boundaries. Sub-horizontal circulation cells are formed in the saltwater entering the aquifer. The penetration of the saltwater can be quantified by a dimensionless buoyancy number that measures the lateral slope of the aquifer relative to freshwater flux. The penetration of the seawater intrusion wedge is controlled more by this slope than by the aquifer thickness and dispersivity. Thus, the slope must be taken into account in order to accurately evaluate seawater intrusion. ?? 2007 Elsevier B.V. All rights reserved.

  13. 3D timelapse analysis of muscle satellite cell motility.

    PubMed

    Siegel, Ashley L; Atchison, Kevin; Fisher, Kevin E; Davis, George E; Cornelison, D D W

    2009-10-01

    Skeletal muscle repair and regeneration requires the activity of satellite cells, a population of myogenic stem cells scattered throughout the tissue and activated to proliferate and differentiate in response to myotrauma or disease. While it seems likely that satellite cells would need to navigate local muscle tissue to reach damaged areas, relatively little data on such motility exist, and most studies have been with immortalized cell lines. We find that primary satellite cells are significantly more motile than myoblast cell lines, and that adhesion to laminin promotes primary cell motility more than fourfold over other substrates. Using timelapse videomicroscopy to assess satellite cell motility on single living myofibers, we have identified a requirement for the laminin-binding integrin alpha 7 beta 1 in satellite cell motility, as well as a role for hepatocyte growth factor in promoting directional persistence. The extensive migratory behavior of satellite cells resident on muscle fibers suggests caution when determining, based on fixed specimens, whether adjacent cells are daughters from the same mother cell. We also observed more persistent long-term contact between individual satellite cells than has been previously supposed, potential cell-cell attractive and repulsive interactions, and migration between host myofibers. Based on such activity, we assayed for expression of "pathfinding" cues, and found that satellite cells express multiple guidance ligands and receptors. Together, these data suggest that satellite cell migration in vivo may be more extensive than currently thought, and could be regulated by combinations of signals, including adhesive haptotaxis, soluble factors, and guidance cues.

  14. 3D Bioprinting a Cell-Laden Bone Matrix for Breast Cancer Metastasis Study.

    PubMed

    Zhou, Xuan; Zhu, Wei; Nowicki, Margaret; Miao, Shida; Cui, Haitao; Holmes, Benjamin; Glazer, Robert I; Zhang, Lijie Grace

    2016-11-09

    Metastasis is one of the deadliest consequences of breast cancer, with bone being one of the primary sites of occurrence. Insufficient 3D biomimetic models currently exist to replicate this process in vitro. In this study, we developed a biomimetic bone matrix using 3D bioprinting technology to investigate the interaction between breast cancer (BrCa) cells and bone stromal cells (fetal osteoblasts and human bone marrow mesenchymal stem cells (MSCs)). A tabletop stereolithography 3D bioprinter was employed to fabricate a series of bone matrices consisting of osteoblasts or MSCs encapsulated in gelatin methacrylate (GelMA) hydrogel with nanocrystalline hydroxyapatite (nHA). When BrCa cells were introduced into the stromal cell-laden bioprinted matrices, we found that the growth of BrCa cells was enhanced by the presence of osteoblasts or MSCs, whereas the proliferation of the osteoblasts or MSCs was inhibited by the BrCa cells. The BrCa cells co-cultured with MSCs or osteoblasts presented increased vascular endothelial growth factor (VEGF) secretion in comparison to that of monocultured BrCa cells. Additionally, the alkaline phosphatase activity of MSCs or osteoblasts was reduced after BrCa cell co-culture. These results demonstrate that the 3D bioprinted matrix, with BrCa cells and bone stromal cells, provides a suitable model with which to study the interactive effects of cells in the context of an artificial bone microenvironment and thus may serve as a valuable tool for the investigation of postmetastatic breast cancer progression in bone.

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

    NASA Astrophysics Data System (ADS)

    Ranjan Gartia, Manas; Hsiao, Austin; Sivaguru, Mayandi; Chen, Yi; Logan Liu, G.

    2011-09-01

    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.

  16. Low intensity pulse ultrasound stimulate chondrocytes growth in a 3-D alginate scaffold through improved porosity and permeability.

    PubMed

    Guo, Gepu; Lu, Lu; Ji, Hongfei; Ma, Yong; Dong, Rui; Tu, Juan; Guo, Xiasheng; Qiu, Yuanyuan; Wu, Junru; Zhang, Dong

    2015-04-01

    A 3-D scaffold culture system has been used to promote in producing functional chondrocytes for repairing damaged cartilage. In the present study, the low intensity pulse ultrasound (LIPUS) (P(-)=0, 0.055, 0.085 and 0.11 MPa) was applied to improve the porosity and permeability of a 3-D alginate scaffold which was beneficial for the nutrition supply and metabolism during cell growth in 3-D alginate scaffold. The porosity and permeability of the scaffold was quantitatively analyzed based on scanning electron microscopy examination and fluorescence image observation. The results suggest that, for the scaffold exposed to LIPUS, its porosity and permeability could be significantly enhanced by the increasing LIPUS amplitude, which might be induced by the microstreaming shear stress generated by ultrasound-driven microbubble oscillations. Furthermore, the assessments of cell proliferation and collagen II expression confirmed that chondrocytes growth could be effectively promoted in 3-D alginate scaffolds treated by LIPUS, because of the improved scaffold porosity and permeability might benefit cell growth space and nutrition supply. It should also be noticed that appropriate LIPUS driving parameters should be adapted to achieve optimized chondrocytes culture effect in 3-D alginate scaffold.

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

    PubMed

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

    2016-08-01

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

  18. 3D Clumped Cell Segmentation Using Curvature Based Seeded Watershed

    PubMed Central

    Atta-Fosu, Thomas; Guo, Weihong; Jeter, Dana; Mizutani, Claudia M.; Stopczynski, Nathan; Sousa-Neves, Rui

    2017-01-01

    Image segmentation is an important process that separates objects from the background and also from each other. Applied to cells, the results can be used for cell counting which is very important in medical diagnosis and treatment, and biological research that is often used by scientists and medical practitioners. Segmenting 3D confocal microscopy images containing cells of different shapes and sizes is still challenging as the nuclei are closely packed. The watershed transform provides an efficient tool in segmenting such nuclei provided a reasonable set of markers can be found in the image. In the presence of low-contrast variation or excessive noise in the given image, the watershed transform leads to over-segmentation (a single object is overly split into multiple objects). The traditional watershed uses the local minima of the input image and will characteristically find multiple minima in one object unless they are specified (marker-controlled watershed). An alternative to using the local minima is by a supervised technique called seeded watershed, which supplies single seeds to replace the minima for the objects. Consequently, the accuracy of a seeded watershed algorithm relies on the accuracy of the predefined seeds. In this paper, we present a segmentation approach based on the geometric morphological properties of the ‘landscape’ using curvatures. The curvatures are computed as the eigenvalues of the Shape matrix, producing accurate seeds that also inherit the original shape of their respective cells. We compare with some popular approaches and show the advantage of the proposed method. PMID:28280723

  19. 3D arrays for high throughput assay of cell migration and electrotaxis.

    PubMed

    Zhao, Sanjun; Gao, Runchi; Devreotes, Peter N; Mogilner, Alex; Zhao, Min

    2013-09-01

    Cell behaviour in 3D environments can be significantly different from those in 2D cultures. With many different 3D matrices being developed and many experimental modalities used to modulate cell behaviour in 3D, it is necessary to develop high throughput techniques to study behaviour in 3D. We report on a 3D array on slide and have adapted this to our electrotaxis chamber, thereby offering a novel approach to quantify cellular responses to electric fields (EFs) in 3D conditions, in different matrices, with different strains of cells, under various field strengths. These developments used Dictyostelium cells to illustrate possible applications and limitations.

  20. Beta-MnO2 3D nanostructures: mineralizer-assisted synthesis, characterization, and growth mechanism.

    PubMed

    Zhou, Fu; Zhao, Xuemei; Yuan, Cunguang; Xu, Hai

    2007-09-01

    The beta-MnO2 three-dimensional (3D) nanostructures were synthesized in large area by a mineralizer-assisted hydrothermal route. KNO3 was introduced as inorganic mineralizer to direct the growth of beta-MnO2 3D nanostructures from Mn(NO3)2 solutions. The samples were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Possible growth mechanism of beta-MnO2 3D nanostructures was proposed based on comparative experiments, indicating that KNO3 mineralizer and the concentration of Mn(NO3)2 solution were the two decisive factors in the fabrication of beta-MnO2 3D nanostructures.

  1. The self-organization of grid cells in 3D.

    PubMed

    Stella, Federico; Treves, Alessandro

    2015-03-30

    Do we expect periodic grid cells to emerge in bats, or perhaps dolphins, exploring a three-dimensional environment? How long will it take? Our self-organizing model, based on ring-rate adaptation, points at a complex answer. The mathematical analysis leads to asymptotic states resembling face centered cubic (FCC) and hexagonal close packed (HCP) crystal structures, which are calculated to be very close to each other in terms of cost function. The simulation of the full model, however, shows that the approach to such asymptotic states involves several sub-processes over distinct time scales. The smoothing of the initially irregular multiple fields of individual units and their arrangement into hexagonal grids over certain best planes are observed to occur relatively quickly, even in large 3D volumes. The correct mutual orientation of the planes, though, and the coordinated arrangement of different units, take a longer time, with the network showing no sign of convergence towards either a pure FCC or HCP ordering.

  2. The self-organization of grid cells in 3D

    PubMed Central

    Stella, Federico; Treves, Alessandro

    2015-01-01

    Do we expect periodic grid cells to emerge in bats, or perhaps dolphins, exploring a three-dimensional environment? How long will it take? Our self-organizing model, based on ring-rate adaptation, points at a complex answer. The mathematical analysis leads to asymptotic states resembling face centered cubic (FCC) and hexagonal close packed (HCP) crystal structures, which are calculated to be very close to each other in terms of cost function. The simulation of the full model, however, shows that the approach to such asymptotic states involves several sub-processes over distinct time scales. The smoothing of the initially irregular multiple fields of individual units and their arrangement into hexagonal grids over certain best planes are observed to occur relatively quickly, even in large 3D volumes. The correct mutual orientation of the planes, though, and the coordinated arrangement of different units, take a longer time, with the network showing no sign of convergence towards either a pure FCC or HCP ordering. DOI: http://dx.doi.org/10.7554/eLife.05913.001 PMID:25821989

  3. 3D dendritic gold nanostructures: seeded growth of a multi-generation fractal architecture.

    PubMed

    Pan, Ming; Xing, Shuangxi; Sun, Ting; Zhou, Wenwen; Sindoro, Melinda; Teo, Hui Hian; Yan, Qingyu; Chen, Hongyu

    2010-10-14

    In this report, we focus on the synthetic challenges for nanoscale 3D fractal architectures, namely the multi-generation growth with control in both size uniformity and colloidal stability; by directing the simultaneous growth of Au and polyaniline on Au seeds, fractal nanoparticles can be achieved with a topology distinctively different from those of spheres, cubes or rods.

  4. Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates

    SciTech Connect

    Nelson, Cassandra E.; Beri, Nina R.; Gardner, Jeffrey G.

    2016-09-21

    Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. Furthermore, we applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.

  5. Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates

    DOE PAGES

    Nelson, Cassandra E.; Beri, Nina R.; Gardner, Jeffrey G.

    2016-09-21

    Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interactionmore » between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. Furthermore, we applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.« less

  6. A 3D in situ cell counter reveals that breast tumor cell (MDA-MB-231) proliferation rate is reduced by the collagen matrix density

    PubMed Central

    Bunaciu, Rodica P.; Yen, Andrew; Wu, Mingming

    2015-01-01

    Many cell types require the biophysical and biochemical cues within the 3D extracellular matrix (ECM) to exhibit their true physiologically relevant behavior. As a result, cell culture platforms have been evolving from traditional 2D petri-dish plates into 3D biomatrices, and there is a need for developing analytic tools to characterize 3D cell culture. The existing cell counting method, using a hemocytometer or coulter counter, requires that cells are suspended in fluids prior to counting. This poses a challenge for 3D cell culture as cells are embedded in a 3D biomatrix. We use a facile 3D cell counting method that overcomes this limitation and allows for in situ cell counting in a 3D cell culture using equipment that is commonly available in a biology lab. Using a breast tumor cell line, MDA-MB-231, as a model system, we demonstrated that MDA-MB-231 cells (1) grow slower within a 3D collagen matrix than on a 2D substrate for an extended growth time (a week) with a comparable, initial cell-to-cell distance, (2) their cell growth rate decreases with the increase of collagen concentration, showing a linear growth rate rather than an exponential growth rate. Further work using flow cytometry showed that the observed growth rate reduction was consistent with the retardation of the transition to S (synthesis) phase in the cell cycle. This work demonstrates the validity of the 3D cell counting method and the importance of cell-ECM interactions in cell proliferation. PMID:25683564

  7. Quantification of substrate and cellular strains in stretchable 3D cell cultures: an experimental and computational framework.

    PubMed

    González-Avalos, P; Mürnseer, M; Deeg, J; Bachmann, A; Spatz, J; Dooley, S; Eils, R; Gladilin, E

    2017-03-07

    The mechanical cell environment is a key regulator of biological processes . In living tissues, cells are embedded into the 3D extracellular matrix and permanently exposed to mechanical forces. Quantification of the cellular strain state in a 3D matrix is therefore the first step towards understanding how physical cues determine single cell and multicellular behaviour. The majority of cell assays are, however, based on 2D cell cultures that lack many essential features of the in vivo cellular environment. Furthermore, nondestructive measurement of substrate and cellular mechanics requires appropriate computational tools for microscopic image analysis and interpretation. Here, we present an experimental and computational framework for generation and quantification of the cellular strain state in 3D cell cultures using a combination of 3D substrate stretcher, multichannel microscopic imaging and computational image analysis. The 3D substrate stretcher enables deformation of living cells embedded in bead-labelled 3D collagen hydrogels. Local substrate and cell deformations are determined by tracking displacement of fluorescent beads with subsequent finite element interpolation of cell strains over a tetrahedral tessellation. In this feasibility study, we debate diverse aspects of deformable 3D culture construction, quantification and evaluation, and present an example of its application for quantitative analysis of a cellular model system based on primary mouse hepatocytes undergoing transforming growth factor (TGF-β) induced epithelial-to-mesenchymal transition.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs.

    PubMed

    Chen, Lei; Xiao, Zhifeng; Meng, Yue; Zhao, Yannan; Han, Jin; Su, Guannan; Chen, Bing; Dai, Jianwu

    2012-02-01

    Three-dimensional (3D) culture could partially simulate in vivo conditions. In this work, we developed a 3D collagen scaffold to investigate cellular properties of MCF-7 cells. The porous scaffolds not only induced the diversification of cell morphologies but also extended cell proliferation. The expression of pro-angiogenic growth factors and the transcriptions of matrix metalloproteinases (MMPs) were significantly increased in cells cultured in 3D collagen scaffolds. In addition, 3D collagen scaffolds could generate a cell population with the properties of cancer stem cells (CSCs). The upregulation of EMT markers and the downregulation of the epithelial cell marker were observed in cells cultured in collagen scaffolds. The expression of stem cell markers, including OCT4A and SOX2, and breast cancer stem cell signatures, including SOX4, JAG1 and CD49F, was significantly unregulated in 3D collagen scaffolds. The proportion of cells with CSC-like CD44(+)/CD24(-/low) phenotype was notably increased. High-level expression of CSC-associated properties of MCF-7 cells cultured in 3D was further confirmed by high tumorigenicity in vivo. Moreover, xenografts with 3D cells formed larger tumors. The properties of MCF-7 cells in 3D may have partially simulated their in vivo behaviors. Thus, 3D collagen scaffolds might provide a useful platform for anti-cancer therapeutics and CSC research.

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Zonca, Michael R., Jr.

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

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

    NASA Astrophysics Data System (ADS)

    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.

  13. 3D Printed Trileaflet Valve Conduits Using Biological Hydrogels and Human Valve Interstitial Cells

    PubMed Central

    Duan, Bin; Kapetanovic, Edi; Hockaday, Laura A.; Butcher, Jonathan T.

    2014-01-01

    Tissue engineering has great potential to provide a functional de novo living valve replacement capable of integration with host tissue and growth. Among various valve conduit fabrication techniques, 3D bioprinting enables deposition of cells and hydrogels into 3D constructs with anatomical geometry and heterogeneous mechanical properties. Successful translation of this approach is however constrained by the dearth of printable and biocompatible hydrogel materials. Furthermore, it is not known how human valve cells respond to these printed environments. In this study, we develop 3D printable formulations of hybrid hydrogels based on methacrylated hyaluronic acid (Me-HA) and methacrylated gelatin (Me-Gel), and utilize them to bioprint heart valve conduits containing encapsulated human aortic valvular interstitial cells (HAVIC). Increasing Me-Gel concentration resulted in lower stiffness and higher viscosity, facilitated cell spreading, and better maintained HAVIC fibroblastic phenotype. Bioprinting accuracy was dependent upon the relative concentrations of Me-Gel and Me-HA, but when optimized enabled the fabrication of a trileaflet valve shape accurate to the original design. HAVIC encapsulated within bioprinted heart valves maintained high viability, and remodeled the initial matrix by depositing collagen and glyosaminoglycans. These findings represent the first rational design of bioprinted trileaflet valve hydrogels that regulate encapsulated human VIC behavior. The use of anatomically accurate living valve scaffolds through bioprinting may accelerate our understanding of physiological valve cell interactions and our progress towards de novo living valve replacements. PMID:24334142

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

    PubMed Central

    Gledhill, Karl; Guo, Zongyou; Umegaki-Arao, Noriko; Higgins, Claire A.; Itoh, Munenari; Christiano, Angela M.

    2015-01-01

    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. PMID:26308443

  15. Peptide hydrogelation and cell encapsulation for 3D culture of MCF-7 breast cancer cells.

    PubMed

    Huang, Hongzhou; Ding, Ying; Sun, Xiuzhi S; Nguyen, Thu A

    2013-01-01

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

  16. The application of low shear modeled microgravity to 3-D cell biology and tissue engineering.

    PubMed

    Navran, Stephen

    2008-01-01

    The practice of cell culture has been virtually unchanged for 100 years. Until recently, life scientists have had to content themselves with two-dimensional cell culture technology. Clearly, living creatures are not constructed in two dimensions and thus it has become widely recognized that in vitro culture systems must become three dimensional to correctly model in vivo biology. Attempts to modify conventional 2-D culture technology to accommodate 3-D cell growth such as embedding cells in extracellular matrix have demonstrated the superiority of concept. Nevertheless, there are serious drawbacks to this approach including limited mass transport and lack of scalability. Recently, a new cell culture technology developed at NASA to study the effects of microgravity on cells has emerged to solve many of the problems of 3-D cell culture. The technology, the Rotating Wall Vessel (RWV) is a single axis clinostat consisting of a fluid-filled, cylindrical, horizontally rotating culture vessel. Cells placed in this environment are suspended by the resolution of the gravitational, centrifugal and Coriolis forces with extremely low mechanical shear. These conditions, which have been called "low shear modeled microgravity", enable cells to assemble into tissue-like aggregates with high mass transport of nutrients, oxygen and wastes. Examples of the use of the RWV for basic cell biology research and tissue engineering applications are discussed.

  17. Hydrodynamic growth experiments with the 3-D, “native-roughness” modulations on NIF

    NASA Astrophysics Data System (ADS)

    Smalyuk, V. A.; Weber, S. V.; Casey, D.; Clark, D. S.; Coppari, F.; Field, J. E.; Haan, S. W.; Hammel, B. A.; Hamza, A.; Hsing, W.; Landen, O.; Nikroo, A.; Robey, H. F.; Weber, C. R.

    2016-05-01

    Hydrodynamic instability growth experiments with threedimensional (3-D) surface-roughness modulations were performed on plastic (CH) shell spherical implosions at the National Ignition Facility (NIF). The initial capsule outer-surface roughness was similar to the standard specifications (“native roughness”) used in a majority of implosions on NIF. At a convergence ratio of ∼3, the measured tent modulations were close to those predicted by 3-D simulations (within ∼15-20%), while measured 3-D, broadband modulations were ∼3-4 times larger than those simulated based on the growth of the known imposed initial surface modulations. One of the hypotheses to explain the results is based on the increased instability amplitudes due to modulations of the oxygen content in the bulk of the capsule. These new experiments results have prompted looking for ways to reduce UV light exposure during target fabrication.

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

    PubMed

    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.

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

    PubMed Central

    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

  20. Modulus-regulated 3D-cell proliferation in an injectable self-healing hydrogel.

    PubMed

    Li, Yongsan; Zhang, Yingwei; Shi, Feng; Tao, Lei; Wei, Yen; Wang, Xing

    2017-01-01

    Cell therapy has attracted wide attention among researchers in biomaterial and medical areas. As a carrier, hydrogels that could keep high viability of the embedded cells have been developed. However, few researches were conducted on 3D cell proliferation, a key factor for cell therapy, especially after injection. In this study, we demonstrated for the first time the proliferation regulation of the 3D-embedded L929 cells in a modulus-tunable and injectable self-healing hydrogel before and after injection without adding specific growth factor. The cells showed a stiffness-dependent proliferation to grow faster in higher stiffness hydrogels. The proliferating rates of the encapsulated cells before and after injection were quantified, and the shearing force as a possible negative influence factor was discussed, suggesting the both internal property of the hydrogel and injection process are critical for further practical applications. Due to the high operability and good biocompatibility, this injectable self-healing hydrogel can be a promising carrier for cell therapy.

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

    PubMed Central

    Wüst, Silke; Müller, Ralph; Hofmann, Sandra

    2011-01-01

    Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or 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. PMID:24956301

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

    PubMed

    Wüst, Silke; Müller, Ralph; Hofmann, Sandra

    2011-08-04

    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.

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

    ERIC Educational Resources Information Center

    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…

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

    PubMed Central

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

    2016-01-01

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

  5. Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models.

    PubMed

    Tam, Roger Y; Smith, Laura J; Shoichet, Molly S

    2017-03-27

    Conventional cell culture techniques using 2D polystyrene or glass have provided great insight into key biochemical mechanisms responsible for cellular events such as cell proliferation, differentiation, and cell-cell interactions. However, the physical and chemical properties of 2D culture in vitro are dramatically different than those found in the native cellular microenvironment in vivo. Cells grown on 2D substrates differ significantly from those grown in vivo, and this explains, in part, why many promising drug candidates discovered through in vitro drug screening assays fail when they are translated to in vivo animal or human models. To overcome this obstacle, 3D cell culture using biomimetic hydrogels has emerged as an alternative strategy to recapitulate native cell growth in vitro. Hydrogels, which are water-swollen polymers, can be synthetic or naturally derived. Many methods have been developed to control the physical and chemical properties of the hydrogels to match those found in specific tissues. Compared to 2D culture, cells cultured in 3D gels with the appropriate physicochemical cues can behave more like they naturally do in vivo. While conventional hydrogels involve modifications to the bulk material to mimic the static aspects of the cellular microenvironment, recent progress has focused on using more dynamic hydrogels, the chemical and physical properties of which can be altered with external stimuli to better mimic the dynamics of the native cellular microenvironment found in vivo. In this Account, we describe our progress in designing stimuli-responsive, optically transparent hydrogels that can be used as biomimetic extracellular matrices (ECMs) to study cell differentiation and migration in the context of modeling the nervous system and cancer. Specifically, we developed photosensitive agarose and hyaluronic acid hydrogels that are activated by single or two-photon irradiation for biomolecule immobilization at specific volumes within the 3D

  6. Reconstruction and Visualization of Coordinated 3D Cell Migration Based on Optical Flow.

    PubMed

    Kappe, Christopher P; Schütz, Lucas; Gunther, Stefan; Hufnagel, Lars; Lemke, Steffen; Leitte, Heike

    2016-01-01

    Animal development is marked by the repeated reorganization of cells and cell populations, which ultimately determine form and shape of the growing organism. One of the central questions in developmental biology is to understand precisely how cells reorganize, as well as how and to what extent this reorganization is coordinated. While modern microscopes can record video data for every cell during animal development in 3D+t, analyzing these videos remains a major challenge: reconstruction of comprehensive cell tracks turned out to be very demanding especially with decreasing data quality and increasing cell densities. In this paper, we present an analysis pipeline for coordinated cellular motions in developing embryos based on the optical flow of a series of 3D images. We use numerical integration to reconstruct cellular long-term motions in the optical flow of the video, we take care of data validation, and we derive a LIC-based, dense flow visualization for the resulting pathlines. This approach allows us to handle low video quality such as noisy data or poorly separated cells, and it allows the biologists to get a comprehensive understanding of their data by capturing dynamic growth processes in stills. We validate our methods using three videos of growing fruit fly embryos.

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

  8. Microencapsulation of Neuroblastoma Cells and Mesenchymal Stromal Cells in Collagen Microspheres: A 3D Model for Cancer Cell Niche Study

    PubMed Central

    Yeung, Pan; Sin, Hoi Shun; Chan, Shing; Chan, Godfrey Chi Fung; Chan, Barbara Pui

    2015-01-01

    There is a growing trend for researchers to use in vitro 3D models in cancer studies, as they can better recapitulate the complex in vivo situation. And the fact that the progression and development of tumor are closely associated to its stromal microenvironment has been increasingly recognized. The establishment of such tumor supportive niche is vital in understanding tumor progress and metastasis. The mesenchymal origin of many cells residing in the cancer niche provides the rationale to include MSCs in mimicking the niche in neuroblastoma. Here we co-encapsulate and co-culture NBCs and MSCs in a 3D in vitro model and investigate the morphology, growth kinetics and matrix remodeling in the reconstituted stromal environment. Results showed that the incorporation of MSCs in the model lead to accelerated growth of cancer cells as well as recapitulation of at least partially the tumor microenvironment in vivo. The current study therefore demonstrates the feasibility for the collagen microsphere to act as a 3D in vitro cancer model for various topics in cancer studies. PMID:26657086

  9. Comparison of Mesenchymal Stem Cell Source Differentiation Toward Human Pediatric Aortic Valve Interstitial Cells within 3D Engineered Matrices

    PubMed Central

    Duan, Bin; Hockaday, Laura A.; Das, Shoshana; Xu, Charlie

    2015-01-01

    Living tissue-engineered heart valves (TEHV) would be a major benefit for children who require a replacement with the capacity for growth and biological integration. A persistent challenge for TEHV is accessible human cell source(s) that can mimic native valve cell phenotypes and matrix remodeling characteristics that are essential for long-term function. Mesenchymal stem cells derived from bone marrow (BMMSC) or adipose tissue (ADMSC) are intriguing cell sources for TEHV, but they have not been compared with pediatric human aortic valve interstitial cells (pHAVIC) in relevant 3D environments. In this study, we compared the spontaneous and induced multipotency of ADMSC and BMMSC with that of pHAVIC using different induction media within three-dimensional (3D) bioactive hybrid hydrogels with material modulus comparable to that of aortic heart valve leaflets. pHAVIC possessed some multi-lineage differentiation capacity in response to induction media, but limited to the earliest stages and much less potent than either ADMSC or BMMSC. ADMSC expressed cell phenotype markers more similar to pHAVIC when conditioned in basic fibroblast growth factor (bFGF) containing HAVIC growth medium, while BMMSC generally expressed similar extracellular matrix remodeling characteristics to pHAVIC. Finally, we covalently attached bFGF to PEG monoacrylate linkers and further covalently immobilized in the 3D hybrid hydrogels. Immobilized bFGF upregulated vimentin expression and promoted the fibroblastic differentiation of pHAVIC, ADMSC, and BMMSC. These findings suggest that stem cells retain a heightened capacity for osteogenic differentiation in 3D culture, but can be shifted toward fibroblast differentiation through matrix tethering of bFGF. Such a strategy is likely important for utilizing stem cell sources in heart valve tissue engineering applications. PMID:25594437

  10. QSAR and 3D QSAR of inhibitors of the epidermal growth factor receptor

    NASA Astrophysics Data System (ADS)

    Pinto-Bazurco, Mariano; Tsakovska, Ivanka; Pajeva, Ilza

    This article reports quantitative structure-activity relationships (QSAR) and 3D QSAR models of 134 structurally diverse inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase. Free-Wilson analysis was used to derive the QSAR model. It identified the substituents in aniline, the polycyclic system, and the substituents at the 6- and 7-positions of the polycyclic system as the most important structural features. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used in the 3D QSAR modeling. The steric and electrostatic interactions proved the most important for the inhibitory effect. Both QSAR and 3D QSAR models led to consistent results. On the basis of the statistically significant models, new structures were proposed and their inhibitory activities were predicted.

  11. Filopodia in cell adhesion, 3D migration and cancer cell invasion.

    PubMed

    Jacquemet, Guillaume; Hamidi, Hellyeh; Ivaska, Johanna

    2015-10-01

    This review discusses recent advances in our understanding of the role filopodia and filopodia-like structures in cell adhesion and three dimensional (3D) cell migration both in vitro and in vivo. In particular, we focus on recent advances demonstrating that filopodia are involved in substrate tethering and environment sensing in vivo. We further discuss the emerging role of filopodia and filopodial proteins in tumor dissemination as mounting in vitro, in vivo and clinical evidence suggest that filopodia drive cancer cell invasion and highlight filopodia proteins as attractive therapeutic targets. Finally, we outline outstanding questions that remain to be addressed to elucidate the role of filopodia during 3D cell migration.

  12. Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions.

    PubMed

    Altmann, Brigitte; Löchner, Anne; Swain, Michael; Kohal, Ralf-Joachim; Giselbrecht, Stefan; Gottwald, Eric; Steinberg, Thorsten; Tomakidi, Pascal

    2014-03-01

    As information on osteoblast mechanosensitivity response to biomechanical cues in three-dimensional (3D) in vitro microenvironments is sparse, the present study compared morphogenesis of primary human alveolar bone osteoblasts (PHABO) under microchip-based 3D-static conditions, and 3D-fluid flow-mediated biomechanical stimulation in perfusion bioreactors. Discrimination of the respective microenvironment by differential morphogenesis was evident from fluid flow-induced PHABO reorganization into rotund bony microtissue, comprising more densely packed multicellular 3D-aggregates, while viability of microtissues was flow rate dependent. Time-lapse microscopy and simple modeling of biomechanical conditions revealed that physiologically relevant fluid flow-mediated PHABO stimulation was associated with formation of mulberry-like PHABO aggregates within the first 24 h. Differential extracellular matrix deposition patterns and gene expression modulation in PHABO aggregates at day 7 further indicates progressive osteoblast differentiation exclusively in perfusion culture-developed bony microtissues. The results of our study strongly suggest PHABO morphogenesis as discriminator of microenvironmental growth conditions, which in case of the microfluidic 3D microchip-bioreactor are substantiated by triggering in vitro bone microtissue formation concomitant with progressive osteoblastic differentiation. Such microtissue outcomes provide unique insight for mechanobiological studies in response to biomechanical fluid flow cues, and clinically appear promising for in vitro PHABO preconditioning, enabling innovative bone augmentation procedures.

  13. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

    PubMed

    Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

    2015-11-01

    Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.

  14. Guiding Cell Attachment in 3D Microscaffolds Selectively Functionalized with Two Distinct Adhesion Proteins.

    PubMed

    Richter, Benjamin; Hahn, Vincent; Bertels, Sarah; Claus, Tanja K; Wegener, Martin; Delaittre, Guillaume; Barner-Kowollik, Christopher; Bastmeyer, Martin

    2017-02-01

    The combination of three different photoresists into a single direct laser written 3D microscaffold permits functionalization with two bioactive full-length proteins. The cell-instructive microscaffolds consist of a passivating framework equipped with light activatable constituents featuring distinct protein-binding properties. This allows directed cell attachment of epithelial or fibroblast cells in 3D.

  15. Ezrin and BCAR1/p130Cas mediate breast cancer growth as 3-D spheroids.

    PubMed

    Konstantinovsky, Sophya; Davidson, Ben; Reich, Reuven

    2012-08-01

    CAS proteins and Ezrin, Radixin, Moesin (ERM) family members act as intracellular scaffolds and are involved in interactions with the cytoskeleton, respectively. Both protein families have previously been associated with metastasis and poor prognosis in cancer. Our group recently reported on the overexpression of EZR/VIL2 and BCAR1 and their protein products in breast carcinoma effusions compared to primary breast carcinoma. In the present study, the role of these two proteins was studied in semi-normal MCF10A cells and metastatic MDA-MB-231 breast carcinoma cells cultured in tri-dimensional (3-D) conditions that were hypothesized to reproduce the in vivo conditions of breast cancer metastasis. MCF10A cells formed spheroid-shaped colonies without any Matrigel invasion, while MDA-MB-231 cells displayed an invasive phenotype and showed satellite projections that bridged multiple cell colonies in 3-D culture. E-cadherin was expressed in MCF10A, but not in MDA-MB-231 cells. The temporal expression of ezrin and BCAR1/p130Cas at the mRNA and protein level differed in the two cell lines upon 3-D culturing on Matrigel. Upregulation of BCAR1/p130cas was observed in the transition of MDA-MB-231 from attached to detached culture. Silencing of Ezrin and p130Cas in MDA-MB-231 cells by short hairpin RNA resulted in decreased invasive potential, and p130Cas silencing further resulted in smaller spheroid/colony formation. Our data show that MCF10A and MDA-MB-231 cells differ in their ability to form spheroids, in expression of E-cadherin and in the expression of Ezrin and BCAR1/p130Cas in 3-D cultures on Matrigel, suggesting a role in tumor progression in breast carcinoma.

  16. Growth trajectories of the human fetal brain tissues estimated from 3D reconstructed in utero MRI

    PubMed Central

    Scott, Julia A.; Habas, Piotr A.; Kim, Kio; Rajagopalan, Vidya; Hamzelou, Kia S.; Corbett-Detig, James M.; Barkovich, A. James; Glenn, Orit A.; Studholme, Colin

    2012-01-01

    In the latter half of gestation (20 to 40 gestational weeks), human brain growth accelerates in conjunction with cortical folding and the deceleration of ventricular zone progenitor cell proliferation. These processes are reflected in changes in the volume of respective fetal tissue zones. Thus far, growth trajectories of the fetal tissue zones have been extracted primarily from 2D measurements on histological sections and magnetic resonance imaging (MRI). In this study, the volumes of major fetal zones—cortical plate (CP), subplate and intermediate zone (SP+IZ), germinal matrix (GMAT), deep gray nuclei (DG), and ventricles (VENT)—are calculated from automatic segmentation of motion-corrected, 3D reconstructed MRI. We analyzed 48 T2-weighted MRI scans from 39 normally developing fetuses in utero between 20.57 and 31.14 gestational weeks (GW). The supratentorial volume (STV) increased linearly at a rate of 15.22% per week. The SP+IZ (14.75% per week) and DG (15.56% per week) volumes increased at similar rates. The CP increased at a greater relative rate (18.00% per week), while the VENT (9.18% per week) changed more slowly. Therefore, CP increased as a fraction of STV and the VENT fraction declined. The total GMAT volume slightly increased then decreased after 25 GW. We did not detect volumetric sexual dimorphisms or total hemispheric volume asymmetries, which may emerge later in gestation. Further application of the automated fetal brain segmentation to later gestational ages will bridge the gap between volumetric studies of premature brain development and normal brain development in utero. PMID:21530634

  17. Growth trajectories of the human fetal brain tissues estimated from 3D reconstructed in utero MRI.

    PubMed

    Scott, Julia A; Habas, Piotr A; Kim, Kio; Rajagopalan, Vidya; Hamzelou, Kia S; Corbett-Detig, James M; Barkovich, A James; Glenn, Orit A; Studholme, Colin

    2011-08-01

    In the latter half of gestation (20-40 gestational weeks), human brain growth accelerates in conjunction with cortical folding and the deceleration of ventricular zone progenitor cell proliferation. These processes are reflected in changes in the volume of respective fetal tissue zones. Thus far, growth trajectories of the fetal tissue zones have been extracted primarily from 2D measurements on histological sections and magnetic resonance imaging (MRI). In this study, the volumes of major fetal zones-cortical plate (CP), subplate and intermediate zone (SP+IZ), germinal matrix (GMAT), deep gray nuclei (DG), and ventricles (VENT)--are calculated from automatic segmentation of motion-corrected, 3D reconstructed MRI. We analyzed 48 T2-weighted MRI scans from 39 normally developing fetuses in utero between 20.57 and 31.14 gestational weeks (GW). The supratentorial volume (STV) increased linearly at a rate of 15.22% per week. The SP+IZ (14.75% per week) and DG (15.56% per week) volumes increased at similar rates. The CP increased at a greater relative rate (18.00% per week), while the VENT (9.18% per week) changed more slowly. Therefore, CP increased as a fraction of STV and the VENT fraction declined. The total GMAT volume slightly increased then decreased after 25 GW. We did not detect volumetric sexual dimorphisms or total hemispheric volume asymmetries, which may emerge later in gestation. Further application of the automated fetal brain segmentation to later gestational ages will bridge the gap between volumetric studies of premature brain development and normal brain development in utero.

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

    PubMed

    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

  19. Confined Growth of Metal Nanoparticles Within 3D DNA Origami Molds.

    PubMed

    Sun, Wei; Shen, Jie

    2017-01-01

    Manufacturing prescribed shaped metal nanoparticles promises emerging applications in plasmonics, energy, and disease diagnosis. The key to the shape-controllable synthesis is generating local environments encoded with prescribed geometrical information. Here, we describe a general strategy that uses 3D self-assembled DNA origami as mold to confine the casting growth of metal nanoparticle. By transferring the shape information from DNA cavities to metal nanoparticles, metal nanoparticles with prescribed shapes, dimensions, and surface binding features could be rationally designed and synthesized.

  20. Fabrication of microfluidic system for the assessment of cell migration on 3D micropatterned substrates.

    PubMed

    Lee, Eun-Joong; Hwang, Chang-Mo; Baek, Dong-Hyun; Lee, Sang-Hoon

    2009-01-01

    Cell migration and proliferation are major process in wound healing, cancer metastasis and organogenesis during development. Many cells are related to recovery process of wound. Especially, fibroblasts act an important role in wound healing. Various cytokines such as platelet derived growth factor (PDGF) can induce fibroblast migration and widely studied to investigate the cell response under controlled cytokine microenvironments during wound healing. In real tissue healing process, cell microenvironments change with tissue types and anatomical characteristics of organs. With microfluidic system, we tried to mimic the natural microenvironment of wound healing, with gradient of PDGF, a fibroblast migration inducing cytokine, and patterned substrate with different orientation to PDGF gradient. Fibroblasts cultured in PDGF gradient micro fluidic chip showed cell migration under various micro environmental gradient conditions. Cells were cultured under PDGF gradient condition and different substrate pattern. Mouse fibroblast L929 cells were cultured in the microfluidic gradient. The results showed that most cells migrated along the substrate topological patterns under high concentration of PDGF. We developed long range sustaining micro fluidic channel and could analyze cell migration along the gradient of PDGF. Also, the cell migration on patterned extracellular environment shows that cells migrate along the extracellular 3D pattern rather than directly along the cytokine gradient when the pattern height is less than 1 microm. In this study, we could demonstrate that the extracellular pattern is more dominant to cell migration in combination with cytokine gradient in the wounded tissue when the environmental cues are 20 microm.

  1. Silk scaffolds connected with different naturally occurring biomaterials for prostate cancer cell cultivation in 3D.

    PubMed

    Bäcker, Anne; Erhardt, Olga; Wietbrock, Lukas; Schel, Natalia; Göppert, Bettina; Dirschka, Marian; Abaffy, Paul; Sollich, Thomas; Cecilia, Angelica; Gruhl, Friederike J

    2017-02-01

    In the present work, different biopolymer blend scaffolds based on the silk protein fibroin from Bombyx mori (BM) were prepared via freeze-drying method. The chemical, structural, and mechanical properties of the three dimensional (3D) porous silk fibroin (SF) composite scaffolds of gelatin, collagen, and chitosan as well as SF from Antheraea pernyi (AP) and the recombinant spider silk protein spidroin (SSP1) have been systematically investigated, followed by cell culture experiments with epithelial prostate cancer cells (LNCaP) up to 14 days. Compared to the pure SF scaffold of BM, the blend scaffolds differ in porous morphology, elasticity, swelling behavior, and biochemical composition. The new composite scaffold with SSP1 showed an increased swelling degree and soft tissue like elastic properties. Whereas, in vitro cultivation of LNCaP cells demonstrated an increased growth behavior and spheroid formation within chitosan blended scaffolds based on its remarkable porosity, which supports nutrient supply matrix. Results of this study suggest that silk fibroin matrices are sufficient and certain SF composite scaffolds even improve 3D cell cultivation for prostate cancer research compared to matrices based on pure biomaterials or synthetic polymers.

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

    NASA Astrophysics Data System (ADS)

    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.

  3. A porous 3D cell culture micro device for cell migration study.

    PubMed

    Ma, Liang; Zhou, Changchun; Lin, Biaoyang; Li, Wei

    2010-08-01

    Cell migration under chemoattractant is an important biological step in cancer metastasis that causes the spread of malignant tumor cells. Porous polymeric materials are widely used to mimic the extracellular matrix (ECM) environment for applications such as three dimensional (3D) cell culturing and tissue engineering. In this paper we report a novel 3D cell culture device based on porous polymeric material to study cancer migration. We fabricated a porous channel on a polymeric chip using a selective ultrasonic foaming method. We demonstrate that a chemical concentration gradient could be established through the porous channel due to the slow diffusion process. We show that significant cell migration could be observed through the porous channel within 1-2 weeks of cell culturing when metastatic M4A4-GFP breast cancer cells were induced by 20% fetal bovine serum (FBS).We also developed a mathematical model to evaluate the diffusivity and concentration gradient through the fabricated porous structure.

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

    PubMed

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

    2016-07-01

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

  5. The study of craniofacial growth patterns using 3D laser scanning and geometric morphometrics

    NASA Astrophysics Data System (ADS)

    Friess, Martin

    2006-02-01

    Throughout childhood, braincase and face grow at different rates and therefore exhibit variable proportions and positions relative to each other. Our understanding of the direction and magnitude of these growth patterns is crucial for many ergonomic applications and can be improved by advanced 3D morphometrics. The purpose of this study is to investigate this known growth allometry using 3D imaging techniques. The geometry of the head and face of 840 children, aged 2 to 19, was captured with a laser surface scanner and analyzed statistically. From each scan, 18 landmarks were extracted and registered using General Procrustes Analysis (GPA). GPA eliminates unwanted variation due to position, orientation and scale by applying a least-squares superimposition algorithm to individual landmark configurations. This approach provides the necessary normalization for the study of differences in size, shape, and their interaction (allometry). The results show that throughout adolescence, boys and girls follow a different growth trajectory, leading to marked differences not only in size but also in shape, most notably in relative proportions of the braincase. These differences can be observed during early childhood, but become most noticeable after the age of 13 years, when craniofacial growth in girls slows down significantly, whereas growth in boys continues for at least 3 more years.

  6. Microfluidic assay of endothelial cell migration in 3D interpenetrating polymer semi-network HA-Collagen hydrogel.

    PubMed

    Jeong, Gi Seok; Kwon, Gu Han; Kang, Ah Ran; Jung, Bo Young; Park, Yongdoo; Chung, Seok; Lee, Sang-Hoon

    2011-08-01

    Cell migration through the extracellular matrix (ECM) is one of the key features for physiological and pathological processes such as angiogenesis, cancer metastasis, and wound healing. In particular, the quantitative assay of endothelial cell migration under the well-defined three dimensional (3D) microenvironment is important to analyze the angiogenesis mechanism. In this study, we report a microfluidic assay of endothelial cell sprouting and migration into an interpenetrating polymer semi-network HA-Collagen (SIPNs CH) hydrogel as ECM providing an enhanced in vivo mimicking 3D microenvironment to cells. The microfluidic chip could provide a well-controlled gradient of growth factor to cells, whereas the hydrogel could mimic a well-defined 3D microenvironment in vivo. (In addition/Furthermore, the microfluidic chip gives a well-controlled gradient of growth factor to cells) For this reason, three types of hydrogel, composed of semi-interpenetrating networks of collagen and hyaluronic acid were prepared, and firstly we proved the role of the hydrogel in endothelial cell migration. The diffusion property and swelling ratio of the hydrogel were characterized. It modulated the migration of endothelial cells in quantified manner, also being influenced by additional synthesis of Matrix metalloproteinase(MMP)-sensitive remodeling peptides and Arginine-glycine-lycinee (RGD) cell adhesion peptides. We successfully established a novel cell migration platform by changing major determinants such as ECM material under biochemical synthesis and under growth factor gradients in a microfluidic manner.

  7. Directed 3D Cell Alignment and Elongation in Microengineered Hydrogels

    DTIC Science & Technology

    2010-01-01

    Immortalized human liver carcinoma cells (Hep-G2) were maintained in DMEM supplemented with 10% FBS, 1% P/S and were passaged twice per week. 2.4. Prepolymer ... prepolymer (20% (w/v) in DPBS) was used to coat TMSPMA treated glass slides as previously described [28]. 2.5. Cell encapsulation and micropatterning Cell... prepolymer was pipetted between a TMSPMA coated glass slide and an untreated coverslip (18 mm (w) 18 mm (l)), then exposed to 6.9 mW/cm2 UV light

  8. Preservation of the 3D Phenotype Upon Dispersal of Cultured Cell Spheroids Into Monolayer Cultures.

    PubMed

    Koshkin, Vasilij; Ailles, Laurie E; Liu, Geoffrey; Krylov, Sergey N

    2017-01-01

    In functional cytometric studies, cultured cells are exposed to effectors (e.g., drugs), and the heterogeneity of cell responses are studied using cytometry techniques (e.g., image cytometry). Such studies are difficult to perform on 3D cell cultures. A solution is to disperse 3D clusters and transfer the cells to the 2D state before applying effectors and using cytometry. This approach requires that the lifetime of the 3D phenotype be longer than the duration of the experiment. Here we studied the dynamics of phenotype transformation from 3D to 2D and searched for means of slowing this transformation down in dispersed spheroids of MCF7 cells. We found three functional biomarkers of the 3D phenotype in MCF7 cell spheroids that are absent in the 2D cell culture: (i) the presence of a subpopulation with an elevated drug-expelling capacity; (ii) the presence of a subpopulation with an elevated cytoprotective capacity; and (iii) the accumulation of cells in the G1 phase of the cell cycle. Monitoring these biomarkers in cells transferred from the 3D state to the 2D state revealed their gradual extinction. We found that the combined application of an elevated cell density and thiol-containing medium supplements increased the lifetime of the 3D phenotype by several fold to as long as 96 h. Our results suggest that extending the lifetime of the 3D phenotype in the cells transferred from the 3D state to the 2D state can facilitate detailed functional cytometric studies, such as measurements of population heterogeneity of cytotoxicity, chemosensitivity, and radiosensitivity. J. Cell. Biochem. 118: 154-162, 2017. © 2016 Wiley Periodicals, Inc.

  9. 3-D Microwell Array System for Culturing Virus Infected Tumor Cells

    PubMed Central

    El Assal, Rami; Gurkan, Umut A.; Chen, Pu; Juillard, Franceline; Tocchio, Alessandro; Chinnasamy, Thiruppathiraja; Beauchemin, Chantal; Unluisler, Sebnem; Canikyan, Serli; Holman, Alyssa; Srivatsa, Srikar; Kaye, Kenneth M.; Demirci, Utkan

    2016-01-01

    Cancer cells have been increasingly grown in pharmaceutical research to understand tumorigenesis and develop new therapeutic drugs. Currently, cells are typically grown using two-dimensional (2-D) cell culture approaches, where the native tumor microenvironment is difficult to recapitulate. Thus, one of the main obstacles in oncology is the lack of proper infection models that recount main features present in tumors. In recent years, microtechnology-based platforms have been employed to generate three-dimensional (3-D) models that better mimic the native microenvironment in cell culture. Here, we present an innovative approach to culture Kaposi’s sarcoma-associated herpesvirus (KSHV) infected human B cells in 3-D using a microwell array system. The results demonstrate that the KSHV-infected B cells can be grown up to 15 days in a 3-D culture. Compared with 2-D, cells grown in 3-D had increased numbers of KSHV latency-associated nuclear antigen (LANA) dots, as detected by immunofluorescence microscopy, indicating a higher viral genome copy number. Cells in 3-D also demonstrated a higher rate of lytic reactivation. The 3-D microwell array system has the potential to improve 3-D cell oncology models and allow for better-controlled studies for drug discovery. PMID:28004818

  10. 3-D Microwell Array System for Culturing Virus Infected Tumor Cells.

    PubMed

    El Assal, Rami; Gurkan, Umut A; Chen, Pu; Juillard, Franceline; Tocchio, Alessandro; Chinnasamy, Thiruppathiraja; Beauchemin, Chantal; Unluisler, Sebnem; Canikyan, Serli; Holman, Alyssa; Srivatsa, Srikar; Kaye, Kenneth M; Demirci, Utkan

    2016-12-22

    Cancer cells have been increasingly grown in pharmaceutical research to understand tumorigenesis and develop new therapeutic drugs. Currently, cells are typically grown using two-dimensional (2-D) cell culture approaches, where the native tumor microenvironment is difficult to recapitulate. Thus, one of the main obstacles in oncology is the lack of proper infection models that recount main features present in tumors. In recent years, microtechnology-based platforms have been employed to generate three-dimensional (3-D) models that better mimic the native microenvironment in cell culture. Here, we present an innovative approach to culture Kaposi's sarcoma-associated herpesvirus (KSHV) infected human B cells in 3-D using a microwell array system. The results demonstrate that the KSHV-infected B cells can be grown up to 15 days in a 3-D culture. Compared with 2-D, cells grown in 3-D had increased numbers of KSHV latency-associated nuclear antigen (LANA) dots, as detected by immunofluorescence microscopy, indicating a higher viral genome copy number. Cells in 3-D also demonstrated a higher rate of lytic reactivation. The 3-D microwell array system has the potential to improve 3-D cell oncology models and allow for better-controlled studies for drug discovery.

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

    PubMed

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

    2016-02-09

    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.

  12. 3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients.

    PubMed

    Kamei, Ken-ichiro; Mashimo, Yasumasa; Koyama, Yoshie; Fockenberg, Christopher; Nakashima, Miyuki; Nakajima, Minako; Li, Junjun; Chen, Yong

    2015-04-01

    Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS), the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility, gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors, important regulators of cell/tissue functions in vivo, influence the survival and growth of human embryonic stem cells. Thus, this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    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

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

    DTIC Science & Technology

    2014-08-01

    remark that more features can be added to the model by augmenting the corresponding free energy . 2.2 Transport equations for biomass Given the...density for component i, i = 1, 2, 3. For incompress- ible materials, we enforce ϕ1 + ϕ2 + ϕ3 = 1. (2) 2.1 Thermodynamic free energy We denote the domain...in which the cell resides together with the buffer fluid as Ω. The free energy of this mixture system is proposed as follows, F = ∫ Ω fdx, (3) where f

  17. N-cadherin as a key regulator of collective cell migration in a 3D environment.

    PubMed

    Shih, Wenting; Yamada, Soichiro

    2012-01-01

    Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.

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

    PubMed

    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.

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

    PubMed

    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.

  20. Improving segmentation of 3D touching cell nuclei using flow tracking on surface meshes.

    PubMed

    Li, Gang; Guo, Lei

    2012-01-01

    Automatic segmentation of touching cell nuclei in 3D microscopy images is of great importance in bioimage informatics and computational biology. This paper presents a novel method for improving 3D touching cell nuclei segmentation. Given binary touching nuclei by the method in Li et al. (2007), our method herein consists of several steps: surface mesh reconstruction and curvature information estimation; direction field diffusion on surface meshes; flow tracking on surface meshes; and projection of surface mesh segmentation to volumetric images. The method is validated on both synthesised and real 3D touching cell nuclei images, demonstrating its validity and effectiveness.

  1. Cell-laden 3D bioprinting hydrogel matrix depending on different compositions for soft tissue engineering: Characterization and evaluation.

    PubMed

    Park, Jisun; Lee, Sang Jin; Chung, Solchan; Lee, Jun Hee; Kim, Wan Doo; Lee, Jae Young; Park, Su A

    2017-02-01

    Cell-printing techniques that can construct three-dimensional (3D) structures with biocompatible materials and cells are of great interest for various biomedical applications, such as tissue engineering and drug-screening studies. For successful cell-printing with cells, bioinks are critical for both the processability of printing and the viability of printed cells. However, the influence of composition on 3D bio-printing with cells has not been well explored. In this study, we investigated different compositions of alginate bioinks by varying the concentrations of high molecular weight alginate (High Alg) and low molecular weight alginate (Low Alg). Bioinks of 3wt% alginate containing High Alg alone or a 1:2 (Low Alg:High Alg) composite allowed for the construction of 3D scaffolds with good processability and shapes. Cell-printing with fibroblasts and in vitro culture studies revealed good viability and growth of the printed cells after up to 7days of culture. Bioinks prepared with High and Low Alg at a 2:1 ratio exhibited better cell growth compared with those of other compositions. This study progresses the design and applications of alginate-based bioinks for cell-printing platforms in soft tissue engineering.

  2. Micro-well arrays for 3D shape control and high resolution analysis of single cells.

    PubMed

    Ochsner, Mirjam; Dusseiller, Marc R; Grandin, H Michelle; Luna-Morris, Sheila; Textor, Marcus; Vogel, Viola; Smith, Michael L

    2007-08-01

    In addition to rigidity, matrix composition, and cell shape, dimensionality is now considered an important property of the cell microenvironment which directs cell behavior. However, available tools for cell culture in two-dimensional (2D) versus three-dimensional (3D) environments are difficult to compare, and no tools exist which provide 3D shape control of single cells. We developed polydimethylsiloxane (PDMS) substrates for the culture of single cells in 3D arrays which are compatible with high-resolution microscopy. Cell adhesion was limited to within microwells by passivation of the flat upper surface through 'wet-printing' of a non-fouling polymer and backfilling of the wells with specific adhesive proteins or lipid bilayers. Endothelial cells constrained within microwells were viable, and intracellular features could be imaged with high resolution objectives. Finally, phalloidin staining of actin stress fibers showed that the cytoskeleton of cells in microwells was 3D and not limited to the cell-substrate interface. Thus, microwells can be used to produce microenvironments for large numbers of single cells with 3D shape control and can be added to a repertoire of tools which are ever more sought after for both fundamental biological studies as well as high throughput cell screening assays.

  3. 3D cell-printing of large-volume tissues: Application to ear regeneration.

    PubMed

    Lee, Jung-Seob; Kim, Byung Soo; Seo, Dong Hwan; Park, Jeong Hun; Cho, Dong-Woo

    2017-01-17

    The three-dimensional (3D) printing of large-volume cells, printed in a clinically relevant size, is one of the most important challenges in the field of tissue engineering. However, few studies have reported the fabrication of large-volume cell-printed constructs (LCCs). To create LCCs, appropriate fabrication conditions should be established: factors involved include fabrication time, residence time, and temperature control of the cell-laden hydrogel in the syringe to ensure high cell viability and functionality. The prolonged time required for 3D printing of LCCs can reduce cell viability and result in insufficient functionality of the construct, because the cells are exposed to a harsh environment during the printing process. In this regard, we present an advanced 3D cell-printing system composed of a clean air workstation, humidifier, and Peltier system, which provides a suitable printing environment for production of LCCs with high cell viability. We confirmed that the advanced 3D cell-printing system was capable of providing enhanced printability of hydrogels and fabricating an ear-shaped LCC with high cell viability. In vivo results for the ear-shaped LCC also showed that printed chondrocytes proliferated sufficiently and differentiated into cartilage tissue. Thus, we conclude that the advanced 3D cell-printing system is a versatile tool to create cell-printed constructs for the generation of large-volume tissues.

  4. Rapid microwave-assisted growth of silver nanoparticles on 3D graphene networks for supercapacitor application.

    PubMed

    Khamlich, S; Khamliche, T; Dhlamini, M S; Khenfouch, M; Mothudi, B M; Maaza, M

    2017-05-01

    Silver nanoparticles (AgNPs) grown on a three dimensional (3d) graphene networks (GNs) has been successfully prepared by an efficient and rapid microwave-assisted growth process to form GNs/AgNPs nanocomposite electrode materials for supercapacitor application. The 3d nature of the used GNs offers a unique architecture, which creates an efficient conduction networks and maximum utilization of space and interface, and acts as a conductive layer for the deposited AgNPs. The electrochemical performances of the fabricated electrode were evaluated by cyclic voltammetry (CV), galvanostatic charge/discharge and electrochemical impedance spectroscopy (EIS) tests. Specifically, the optimal GNs/AgNPs nanocomposite exhibits remarkable performances with a high specific capacitance of 528Fg(-1) at a current density of 1Ag(-1) and excellent capacitance retention of ∼93% after 3000cycles. Moreover, this microwave-assisted growth strategy of AgNPs is simple and effective, which could be extended to the construction of other three dimensional graphene based metallic composites for energy storage and conversion applications.

  5. In situ growth rates of deep-water octocorals determined from 3D photogrammetric reconstructions

    NASA Astrophysics Data System (ADS)

    Bennecke, Swaantje; Kwasnitschka, Tom; Metaxas, Anna; Dullo, Wolf-Christian

    2016-12-01

    Growth rates of deep-water corals provide important information on the recovery potential of these ecosystems, for example from fisheries-induced impacts. Here, we present in situ growth dynamics that are currently largely unknown for deep-water octocorals, calculated by applying a non-destructive method. Videos of a boulder harbouring multiple colonies of Paragorgia arborea and Primnoa resedaeformis in the Northeast Channel Coral Conservation Area at the entrance to the Gulf of Maine at 863 m depth were collected in 2006, 2010 and 2014. Photogrammetric reconstructions of the boulder and the fauna yielded georeferenced 3D models for all sampling years. Repeated measurements of total length and cross-sectional area of the same colonies allowed the observation of growth dynamics. Growth rates of total length of Paragorgia arborea decreased over time with higher rates between 2006 and 2010 than between 2010 and 2014, while growth rates of cross-sectional area remained comparatively constant. A general trend of decreasing growth rates of total length with size of the coral colony was documented. While no growth was observed for the largest colony (165 cm in length) between 2010 and 2014, a colony 50-65 cm in length grew 3.7 cm yr-1 between 2006 and 2010. Minimum growth rates of 1.6-2.7 cm yr-1 were estimated for two recruits (<23 cm in 2014) of Primnoa resedaeformis. We successfully extracted biologically meaningful data from photogrammetric models and present the first in situ growth rates for these coral species in the Northwest Atlantic.

  6. Osteoblasts and Bone Marrow Mesenchymal Stromal Cells Control Hematopoietic Stem Cell Migration and Proliferation in 3D In Vitro Model

    PubMed Central

    de Barros, Ana Paula D. N.; Takiya, Christina M.; Garzoni, Luciana R.; Leal-Ferreira, Mona Lisa; Dutra, Hélio S.; Chiarini, Luciana B.; Meirelles, Maria Nazareth; Borojevic, Radovan; Rossi, Maria Isabel D.

    2010-01-01

    Background Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. Methodology/Principal Findings A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34+ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34+ cells was decreased. Conclusions/Significance Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation. PMID:20161704

  7. Thrust fault growth within accretionary wedges: New Insights from 3D seismic reflection data

    NASA Astrophysics Data System (ADS)

    Orme, H.; Bell, R. E.; Jackson, C. A. L.

    2015-12-01

    The shallow parts of subduction megathrust faults are typically thought to be aseismic and incapable of propagating seismic rupture. The 2011 Tohoku-Oki earthquake, however, ruptured all the way to the trench, proving that in some locations rupture can propagate through the accretionary wedge. An improved understanding of the structural character and physical properties of accretionary wedges is therefore crucial to begin to assess why such anomalously shallow seismic rupture occurs. Despite its importance, we know surprisingly little regarding the 3D geometry and kinematics of thrust network development in accretionary prisms, largely due to a lack of 3D seismic reflection data providing high-resolution, 3D images of entire networks. Thus our current understanding is largely underpinned by observations from analogue and numerical modelling, with limited observational data from natural examples. In this contribution we use PSDM, 3D seismic reflection data from the Nankai margin (3D Muroto dataset, available from the UTIG Academic Seismic Portal, Marine Geoscience Data System) to examine how imbricate thrust fault networks evolve during accretionary wedge growth. Previous studies have reported en-echelon thrust fault geometries from the NW part of the dataset, and have related this complex structure to seamount subduction. We unravel the evolution of faults within the protothrust and imbricate thrust zones by interpreting multiple horizons across faults and measuring fault displacement and fold amplitude along-strike; by doing this, we are able to investigate the three dimensional accrual of strain. We document a number of local displacement minima along-strike of faults, suggesting that, the protothrust and imbricate thrusts developed from the linkage of smaller, previously isolated fault segments. We also demonstrate that the majority of faults grew upward from the décollement, although there is some evidence for downward fault propagation. Our observations

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

    SciTech Connect

    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.

  9. Role of dynamin in elongated cell migration in a 3D matrix.

    PubMed

    Lees, Justin G; Gorgani, Nick N; Ammit, Alaina J; McCluskey, Adam; Robinson, Phillip J; O'Neill, Geraldine M

    2015-03-01

    The use of 3-dimensional (3D) collagen gels has yielded new insights into the migratory behaviour of cancer cells. While the large GTPase dynamin has emerged as an important regulator of cancer cell migration and invasion under 2D conditions, its role in 3D migration is unclear. We have used a potent dynamin modulator, a bis-tyrphostin derivative, Ryngo® 1-23, to investigate the role of dynamin in 3D migration in 3 different cell lines. The compound specifically inhibits persistent, elongated 3D migration in U87MG and SMA-560 cells. Treated U87MG cells adopt a rounded morphology that is not due to apoptosis, loss of matrix metalloprotease activity or inhibition of clathrin-mediated endocytosis. Given that Ryngo 1-23 is known to regulate dynamin oligomerisation and actin dynamics at the leading edge, we analysed actin filament distribution. Ryngo 1-23 induced a switch in actin filament organization in 3D cultures resulting in the generation of multiple short actin-rich microspikes. Correlated with the change in actin filament distribution, cells displayed reduced collagen gel contraction. Since acto-myosin force transmission to the extra-cellular matrix underpins persistent, elongated migration, our results suggest that Ryngo 1-23 modulates this process in 3D migration via dynamin-mediated regulation of acto-myosin force transmission to the extra-cellular matrix.

  10. Significant enhancement of power conversion efficiency for dye sensitized solar cell using 1D/3D network nanostructures as photoanodes

    PubMed Central

    Wang, Hao; Wang, Baoyuan; Yu, Jichao; Hu, Yunxia; Xia, Chen; Zhang, Jun; Liu, Rong

    2015-01-01

    The single–crystalline TiO2 nanorod arrays with rutile phase have attracted much attention in the dye sensitized solar cells (DSSCs) applications because of their superior chemical stability, better electron transport properties, higher refractive index and low production cost. However, it suffers from a low surface area as compared with TiO2 nanoparticle films. In order to enlarge the surface area of TiO2 nanorod arrays, the 1D nanorods/3D nanotubes sample was synthesized using a facile two-step hydrothermal process involving hydrothermal growth 1D/3D nanorods and followed by post-etching treatment. In such bi-layer structure, the oriented TiO2 nanorods layer could provide direct pathway for fast electron transportation, and the 3D nanotubes layer offers a higher surface area for dye loading, therefore, the 1D nanorods/3D nanotubes photoanode exhibited faster electron transport and higher surface area than either 1D or 3D nanostructures alone, and an highest efficiency of 7.68% was achieved for the DSSCs based on 1D nanorods/3D nanotubes photoanode with further TiCl4 treatment. PMID:25800933

  11. 3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells

    PubMed Central

    Felts, Richard L.; Narayan, Kedar; Estes, Jacob D.; Shi, Dan; Trubey, Charles M.; Fu, Jing; Hartnell, Lisa M.; Ruthel, Gordon T.; Schneider, Douglas K.; Nagashima, Kunio; Bess, Julian W.; Bavari, Sina; Lowekamp, Bradley C.; Bliss, Donald; Lifson, Jeffrey D.; Subramaniam, Sriram

    2010-01-01

    The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission. PMID:20624966

  12. 3D-printed porous titanium changed femoral head repair growth patterns: osteogenesis and vascularisation in porous titanium.

    PubMed

    Zhu, Wei; Zhao, Yan; Ma, Qi; Wang, Yingjie; Wu, Zhihong; Weng, Xisheng

    2017-04-01

    Osteonecrosis of the femoral head (ONFH) is a major cause of morbidity, and total hip arthroplasty is both traumatic and expensive. Here, we created a gelatine scaffold embedded in uniquely shaped, 3D-printed porous titanium parts, which could attract and promote the proliferation of osteoblasts as well as bone regeneration, as the extracellular matrix (ECM) does in vivo. Interestingly, after hybridisation with platelets, the scaffold exhibited a low yet considerable rate of stable, safe and long-term growth factor release. Additionally, a novel ONFH model was constructed and verified. Scaffolds implanted in this model were found to accelerate bone repair. In conclusion, our scaffold successfully simulates the ECM and considerably accelerates bone regeneration, in which platelets play an indispensable role. We believe that platelets should be emphasised as carriers that may be employed to transport drugs, cytokines and other small molecules to target locations in vivo. In addition, this novel scaffold is a useful material for treating ONFH. An overview of the novel scaffold mimicking the extracellular environment in bone repair. a and b: A gelatine scaffold was cross-linked and freeze-dried within 3D-printed porous titanium. c: Platelets were coated onto the gelatine microscaffold after freeze-drying platelet-rich plasma. d: The microscaffold supported the migration of cells into the titanium pores and their subsequent growth, while the platelets slowly released cell factors, exerting bioactivity.

  13. A 3D model of tumour angiogenic microenvironment to monitor hypoxia effects on cell interactions and cancer stem cell selection.

    PubMed

    Klimkiewicz, Krzysztof; Weglarczyk, Kazimierz; Collet, Guillaume; Paprocka, Maria; Guichard, Alan; Sarna, Michal; Jozkowicz, Alicja; Dulak, Jozef; Sarna, Tadeusz; Grillon, Catherine; Kieda, Claudine

    2017-03-10

    Tumour microenvironment determines the fate of treatments. Reconstitution of tumour conditions is mandatory for alternative in vitro methods devoted to cancer development and the selection of therapeutic strategies. This work describes a 3D model of melanoma growth in its environment. Introducing means to mimic tumour angiogenesis, which turns on tumour progression, the model shows that melanoma tumour spheroids allow reconstitution of solid tumours with stromal cells. Angiogenesis evidenced the differential recruitment of endothelial cells (EC) from early progenitors (EEPCs) to mature ECs. Hypoxia was the key parameter that selected and stabilized melanoma cancer stem like cells (CSCs) phenotype based on aldehyde dehydrogenase expression as the best criterion. The 3D-tumour-model demonstrated the distinct reactivity of ECs toward tumour cells in terms of cellular cross-talk and humoral response. Intra-spheroid cell-to-cell membrane dye exchanges, mediated by intercellular interactions, uncovered the melanoma-to-EEPC cooperation. The resulting changes in tumour milieu were evidenced by the chemokinic composition and hypoxia-related variations in microRNA expression assessed in each cellular component of the spheroids. This method brings new tools to decipher the molecular mechanism of tumour-mediated cell recruitment and for in vitro assessment of therapeutic approaches.

  14. Rapid 3-D delineation of cell nuclei for high-content screening platforms.

    PubMed

    Gertych, Arkadiusz; Ma, Zhaoxuan; Tajbakhsh, Jian; Velásquez-Vacca, Adriana; Knudsen, Beatrice S

    2016-02-01

    High-resolution three-dimensional (3-D) microscopy combined with multiplexing of fluorescent labels allows high-content analysis of large numbers of cell nuclei. The full automation of 3-D screening platforms necessitates image processing algorithms that can accurately and robustly delineate nuclei in images with little to no human intervention. Imaging-based high-content screening was originally developed as a powerful tool for drug discovery. However, cell confluency, complexity of nuclear staining as well as poor contrast between nuclei and background result in slow and unreliable 3-D image processing and therefore negatively affect the performance of studying a drug response. Here, we propose a new method, 3D-RSD, to delineate nuclei by means of 3-D radial symmetries and test it on high-resolution image data of human cancer cells treated by drugs. The nuclei detection performance was evaluated by means of manually generated ground truth from 2351 nuclei (27 confocal stacks). When compared to three other nuclei segmentation methods, 3D-RSD possessed a better true positive rate of 83.3% and F-score of 0.895±0.045 (p-value=0.047). Altogether, 3D-RSD is a method with a very good overall segmentation performance. Furthermore, implementation of radial symmetries offers good processing speed, and makes 3D-RSD less sensitive to staining patterns. In particular, the 3D-RSD method performs well in cell lines, which are often used in imaging-based HCS platforms and are afflicted by nuclear crowding and overlaps that hinder feature extraction.

  15. Rapid 3-D delineation of cell nuclei for high-content screening platforms

    PubMed Central

    Gertych, Arkadiusz; Ma, Zhaoxuan; Tajbakhsh, Jian; Velásquez-Vacca, Adriana; Knudsen, Beatrice S.

    2015-01-01

    High-resolution three-dimensional (3-D) microscopy combined with multiplexing of fluorescent labels allows high-content analysis of large numbers of cell nuclei. The full automation of 3-D screening platforms necessitates image processing algorithms that can accurately and robustly delineate nuclei in images with little to no human intervention. Imaging-based high-content screening was originally developed as a powerful tool for drug discovery. However, cell confluency, complexity of nuclear staining as well as poor contrast between nuclei and background result in slow and unreliable 3-D image processing and therefore negatively affect the performance of studying a drug response. Here, we propose a new method, 3D-RSD, to delineate nuclei by means of 3-D radial symmetries and test it on high-resolution image data of human cancer cells treated by drugs. The nuclei detection performance was evaluated by means of manually generated ground truth from 2351 nuclei (27 confocal stacks). When compared to three other nuclei segmentation methods, 3D-RSD possessed a better true positive rate of 83.3% and F-score of 0.895+/-0.045 (p- value=0.047). Altogether, 3D-RSD is a method with a very good overall segmentation performance. Furthermore, implementation of radial symmetries offers good processing speed, and makes 3D-RSD less sensitive to staining patterns. In particular the 3D-RSG method performs well in cell lines, which are often used in imaging-based HCS platforms and are afflicted by nuclear crowding and overlaps that hinder feature extraction. PMID:25982066

  16. Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.

    PubMed

    Cliffe, Adam; Doupé, David P; Sung, HsinHo; Lim, Isaac Kok Hwee; Ong, Kok Haur; Cheng, Li; Yu, Weimiao

    2017-04-04

    Understanding the mechanisms of collective cell migration is crucial for cancer metastasis, wound healing and many developmental processes. Imaging a migrating cluster in vivo is feasible, but the quantification of individual cell behaviours remains challenging. We have developed an image analysis toolkit, CCMToolKit, to quantify the Drosophila border cell system. In addition to chaotic motion, previous studies reported that the migrating cells are able to migrate in a highly coordinated pattern. We quantify the rotating and running migration modes in 3D while also observing a range of intermediate behaviours. Running mode is driven by cluster external protrusions. Rotating mode is associated with cluster internal cell extensions that could not be easily characterized. Although the cluster moves slower while rotating, individual cells retain their mobility and are in fact slightly more active than in running mode. We also show that individual cells may exchange positions during migration.

  17. 3D inverted colloidal crystals in realistic cell migration assays for drug screening applications.

    PubMed

    da Silva, Joakim; Lautenschläger, Franziska; Kuo, Cheng-Hwa R; Guck, Jochen; Sivaniah, Easan

    2011-12-01

    Screening drugs for their specific impact on cell mechanics, in addition to targeting adhesion and proteolysis, will be important for successfully moderating migration in infiltrative disorders including cancer metastasis. We present 3D inverted colloidal crystals made of hydrogel as a realistic cell migration assay, where the geometry and stiffness can be set independently to mimic the tissue requirements in question. We show the utility of this 3D assay for drug screening purposes, specifically in contrast to conventional 2D migration studies, by surveying the effects of commonly used cytoskeletal toxins that impact cell mechanics. This assay allows studying large cell numbers for good statistics but at single-cell resolution.

  18. Segmentation of whole cells and cell nuclei from 3-D optical microscope images using dynamic programming.

    PubMed

    McCullough, D P; Gudla, P R; Harris, B S; Collins, J A; Meaburn, K J; Nakaya, M A; Yamaguchi, T P; Misteli, T; Lockett, S J

    2008-05-01

    Communications between cells in large part drive tissue development and function, as well as disease-related processes such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue. However, a major restriction on such analyses is the lack of an efficient method that correctly segments each object (cell or nucleus) from 3-D images of an intact tissue specimen. We report a highly reliable and accurate semi-automatic algorithmic method for segmenting fluorescence-labeled cells or nuclei from 3-D tissue images. Segmentation begins with semi-automatic, 2-D object delineation in a user-selected plane, using dynamic programming (DP) to locate the border with an accumulated intensity per unit length greater that any other possible border around the same object. Then the two surfaces of the object in planes above and below the selected plane are found using an algorithm that combines DP and combinatorial searching. Following segmentation, any perceived errors can be interactively corrected. Segmentation accuracy is not significantly affected by intermittent labeling of object surfaces, diffuse surfaces, or spurious signals away from surfaces. The unique strength of the segmentation method was demonstrated on a variety of biological tissue samples where all cells, including irregularly shaped cells, were accurately segmented based on visual inspection.

  19. 3D choroid neovascularization growth prediction based on reaction-diffusion model

    NASA Astrophysics Data System (ADS)

    Zhu, Shuxia; Chen, Xinjian; Shi, Fei; Xiang, Dehui; Zhu, Weifang; Chen, Haoyu

    2016-03-01

    Choroid neovascularization (CNV) is a kind of pathology from the choroid and CNV-related disease is one important cause of vision loss. It is desirable to predict the CNV growth rate so that appropriate treatment can be planned. In this paper, we seek to find a method to predict the growth of CNV based on 3D longitudinal Optical Coherence Tomography (OCT) images. A reaction-diffusion model is proposed for prediction. The method consists of four phases: pre-processing, meshing, CNV growth modeling and prediction. We not only apply the reaction-diffusion model to the disease region, but also take the surrounding tissues into consideration including outer retinal layer, inner retinal layer and choroid layer. The diffusion in these tissues is considered as isotropic. The finite-element-method (FEM) is used to solve the partial differential equations (PDE) in the diffusion model. The curve of CNV growth with treatment are fitted and then we can predict the CNV status in a future time point. The preliminary results demonstrated that our proposed method is accurate and the validity and feasibility of our model is obvious.

  20. A modular segmented-flow platform for 3D cell cultivation.

    PubMed

    Lemke, Karen; Förster, Tobias; Römer, Robert; Quade, Mandy; Wiedemeier, Stefan; Grodrian, Andreas; Gastrock, Gunter

    2015-07-10

    In vitro 3D cell cultivation is promised to equate tissue in vivo more realistically than 2D cell cultivation corresponding to cell-cell and cell-matrix interactions. Therefore, a scalable 3D cultivation platform was developed. This platform, called pipe-based bioreactors (pbb), is based on the segmented-flow technology: aqueous droplets are embedded in a water-immiscible carrier fluid. The droplet volumes range from 60 nL to 20 μL and are used as bioreactors lined up in a tubing like pearls on a string. The modular automated platform basically consists of several modules like a fluid management for a high throughput droplet generation for self-assembly or scaffold-based 3D cell cultivation, a storage module for incubation and storage, and an analysis module for monitoring cell aggregation and proliferation basing on microscopy or photometry. In this report, the self-assembly of murine embryonic stem cells (mESCs) to uniformly sized embryoid bodies (EBs), the cell proliferation, the cell viability as well as the influence on the cell differentiation to cardiomyocytes are described. The integration of a dosage module for medium exchange or agent addition will enable pbb as long-term 3D cell cultivation system for studying stem cell differentiation, e.g. cardiac myogenesis or for diagnostic and therapeutic testing in personalized medicine.

  1. Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells.

    PubMed

    Nandakumar, Vivek; Hansen, Nanna; Glenn, Honor L; Han, Jessica H; Helland, Stephanie; Hernandez, Kathryn; Senechal, Patti; Johnson, Roger H; Bussey, Kimberly J; Meldrum, Deirdre R

    2016-08-09

    The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an 'epigenetic' drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat's differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.

  2. Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells

    PubMed Central

    Nandakumar, Vivek; Hansen, Nanna; Glenn, Honor L.; Han, Jessica H.; Helland, Stephanie; Hernandez, Kathryn; Senechal, Patti; Johnson, Roger H.; Bussey, Kimberly J.; Meldrum, Deirdre R.

    2016-01-01

    The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an ‘epigenetic’ drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat’s differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action. PMID:27503568

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

    PubMed Central

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

    2015-01-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils. PMID:26548801

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

    PubMed

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

    2015-11-09

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  6. Dynamic coupling between fluid flow and vein growth in fractures: a 3D numerical model

    NASA Astrophysics Data System (ADS)

    Schwarz, J.-O.; Enzmann, F.

    2012-04-01

    Fluid flow is one of the main mass transport mechanisms in the Earth's crust and abundant mineral vein networks are important indicators for fluid flow and fluid rock interaction. These systems are dynamic and part of the so called RTM processes (reaction-transport-mechanics). Understanding of mineral vein systems requires coupling of these processes. Here we present a conceptional model for dynamic vein growth of syntaxial, posttectonic veins generated by advective fluid flow and show first results of a numerical model for this scenario. Vein generation requires three processes to occur: (i) fracture generation by mechanical stress e.g. hydro-fracturing, (ii) flow of a supersaturated fluid on that fracture and (iii) crystallization of phase(s) on or in the fracture. 3D synthetic fractures are generated with the SynFrac code (Ogilvie, et al. 2006). Subsequently solutions of the Navier-Stokes equation for this fracture are computed by a computational fluid dynamics code called GeoDict (Wiegmann 2007). Transport (advective and diffusive) of chemical species to growth sites in the fracture and vein growth are computed by a self-written MATLAB script. The numerical model discretizes the wall rock and fracture geometry by volumetric pixels (voxels). Based on this representation, the model computes the three basic functions for vein generation: (a) nucleation, (b) fluid flow with transport of chemical species and (c) growth. The following conditions were chosen for these three modules. Nucleation is heterogeneous and occurs instantaneously at the wall rock/fracture interface. Advective and diffusive flow of a supersaturated fluid and related transport of chemical species occurs according to the computed fluid flow field by GeoDict. Concentration of chemical species at the inflow is constant, representing external fluid buffering. Changes/decrease in the concentration of chemical species occurs only due to vein growth. Growth of nuclei is limited either by transport of

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

    PubMed

    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.

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

    PubMed Central

    Yoon, Jonghee; Kim, Kyoohyun; 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. PMID:26504637

  9. Fibroblast morphogenesis on 3D collagen matrices: the balance between cell clustering and cell migration.

    PubMed

    da Rocha-Azevedo, Bruno; Grinnell, Frederick

    2013-10-01

    Fibroblast clusters have been observed in tissues under a variety of circumstances: in fibrosis and scar, in the formation of hair follicle dermal papilla, and as part of the general process of mesenchymal condensation that takes place during development. Cell clustering has been shown to depend on features of the extracellular matrix, growth factor environment, and mechanisms to stabilize cell-cell interactions. In vitro studies have shown that increasing the potential for cell-cell adhesion relative to cell-substrate adhesion promotes cell clustering. Experimental models to study fibroblast clustering have utilized centrifugation, hanging drops, and substrata with poorly adhesive, soft and mechanically unstable properties. In this review, we summarize work on a new, highly tractable, cell clustering research model in which human fibroblasts are incubated on the surfaces of collagen matrices. Fibroblast clustering occurs under procontractile growth factor conditions (e.g., serum or the serum lipid agonist lysophosphatidic acid) but not under promigratory growth factor conditions (e.g., platelet-derived growth factor) and can be reversed by switching growth factor environments. Cell contraction plays a dual role in clustering to bring cells closer together and to stimulate cells to organize fibronectin into a fibrillar matrix. Binding of fibroblasts to a shared fibronectin fibrillar matrix stabilizes clusters, and fragmentation of the fibrillar matrix occurs when growth factor conditions are switched to promote cell dispersal.

  10. Epidermal growth factor improves the migration and contractility of aged fibroblasts cultured on 3D collagen matrices.

    PubMed

    Kim, Daehwan; Kim, So Young; Mun, Seog Kyun; Rhee, Sangmyung; Kim, Beom Joon

    2015-04-01

    Epidermal growth factor (EGF) plays a critical role in fibroblasts by stimulating the production of collagen and supports cell renewal through the interaction between keratinocytes and fibroblasts. It is well known that the contractile activity of fibroblasts is required for the remodeling of the extracellular matrix (ECM), which contributes to skin elasticity. However, the role of EGF in the contraction of aged fibroblasts under 3-dimensional (3D) culture conditions is not yet fully understood. In the present study, we demonstrated that young fibroblasts spread and proliferated more rapidly than aged fibroblasts under 2-dimensional (2D) culture conditions. Cell migration assay using a nested collagen matrix revealed that the migration of young fibroblasts was also greater than that of aged fibroblasts under 3D culture conditions. However, the addition of recombinant human EGF (rhEGF) resulted in the enhanced migration of aged fibroblasts; the migration rate was similar to that of the young fibroblasts. The aged fibroblasts showed decreased cluster formation compared with the young fibroblasts on the collagen matrix, which was improved by the addition of rhEGF. Furthermore, cell contraction assay revealed that the basal contractility of the aged fibroblasts was lower than that of the young fibroblasts; however, following treatment with rhEGF, the contractility was restored to levels similar or even higher to those of the young fibroblasts. Taken together, our results suggest that rhEGF is a potential renewal agent that acts to improve the migration and contraction of aged fibroblasts more efficiently than young fibroblasts under 3D culture conditions; thus, EGF may have valuable regenerative effects on aged skin.

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Efficient light harvesting with micropatterned 3D pyramidal photoanodes in dye-sensitized solar cells.

    PubMed

    Wooh, Sanghyuk; Yoon, Hyunsik; Jung, Jae-Hyun; Lee, Yong-Gun; Koh, Jai Hyun; Lee, Byoungho; Kang, Yong Soo; Char, Kookheon

    2013-06-11

    3D TiO2 photoanodes in dye-sensitized solar cells (DSCs) are fabricated by the soft lithographic technique for efficient light trapping. An extended strategy to the construction of randomized pyramid structure is developed by the conventional wet-etching of a silicon wafer for low-cost fabrication. Moreover, the futher enhancement of light absorption resulting in photocurrent increase is achieved by combining the 3D photoanode with a conventional scattering layer.

  15. 3D Simulation of the Growth of Alloy Semiconductor Quantum Dots Considering Morphological and Compositional Coupling

    NASA Astrophysics Data System (ADS)

    Guo, Junyan; Zhang, Yong-Wei; Narayanaswamy, Sridhar

    2012-02-01

    Fabrication of quantum dots (QDs) with high density may be realized by self-assembly via heteroepitaxial growth of thin films. Since the electronic and optoelectronic properties of QDs are sensitive to size, morphology, strain and especially composition, it is of great importance to control their composition profiles and morphology, and engineer the strain in them. Since the growth is a dynamic process, which carries out via surface diffusion driven primarily by strain relaxation and entropy change due to chemical intermixing, a strong coupling between morphological and composition evolutions during this process leads to a rather complex dynamics, which has not been fully understood. In this work, a 3-D finite element model is developed, which is capable of modeling the formation, self-assembly and coarsening of hetero-epitaxial alloy islands by considering the coupling of morphological and compositional evolution. Several interesting experimental observations, such as fast coarsening kinetics; asymmetries in composition profile and island shape; lateral motion of alloy islands have been observed in our simulations. Our model predictions have painted a rather complete picture for the entire dynamic evolution during the growth of nanoscale heteroepitaxial islands.

  16. Automatic 3D Cell Analysis in High-Throughput Microarray Using Micropillar and Microwell Chips.

    PubMed

    Lee, Dong Woo; Lee, Moo-Yeal; Ku, Bosung; Nam, Do-Hyun

    2015-10-01

    Area-based and intensity-based 3D cell viability measurement methods are compared in high-throughput screening in order to analyze their effects on the assay results (doubling time and IC50) and their repeatability. Many other 3D cell-based high-throughput screening platforms had been previously introduced, but these had not clearly addressed the effects of the two methods on the assay results and assay repeatability. In this study, the optimal way to analyze 3D cultured cells is achieved by comparing day-to-day data of doubling times and IC50 values obtained from the two methods. In experiments, the U251 cell line is grown in chips. The doubling time, based on the area of the 3D cells, was 27.8 ± 1.8 h (standard deviation: 6.6%) and 27.8 ± 3.8 h (standard deviation: 13.7%) based on the intensity of the 3D cells. The doubling time calculated by area shows a smaller standard deviation than one calculated by intensity. IC50 values calculated by both methods are very similar. The standard deviations of IC50 values for the two methods were within ± 3-fold. The IC50 variations of the 12 compounds were similar regardless of the viability measurement methods and were highly related to the shape of the dose-response curves.

  17. 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

    PubMed

    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.

  18. Statistical analysis of cell migration in 3D using the anisotropic persistent random walk model.

    PubMed

    Wu, Pei-Hsun; Giri, Anjil; Wirtz, Denis

    2015-03-01

    Cell migration through 3D extracellular matrices (ECMs) is crucial to the normal development of tissues and organs and in disease processes, yet adequate analytical tools to characterize 3D migration are lacking. The motility of eukaryotic cells on 2D substrates in the absence of gradients has long been described using persistent random walks (PRWs). Recent work shows that 3D migration is anisotropic and features an exponential mean cell velocity distribution, rendering the PRW model invalid. Here we present a protocol for the analysis of 3D cell motility using the anisotropic PRW model. The software, which is implemented in MATLAB, enables statistical profiling of experimentally observed 2D and 3D cell trajectories, and it extracts the persistence and speed of cells along primary and nonprimary directions and an anisotropic index of migration. Basic computer skills and experience with MATLAB software are recommended for successful use of the protocol. This protocol is highly automated and fast, taking <30 min to analyze trajectory data per biological condition.

  19. 3D texture analysis in renal cell carcinoma tissue image grading.

    PubMed

    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.

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

    PubMed

    Knight, Eleanor; Przyborski, Stefan

    2015-12-01

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

  1. Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds.

    PubMed

    Neofytou, Evgenios A; Chang, Edwin; Patlola, Bhagat; Joubert, Lydia-Marie; Rajadas, Jayakumar; Gambhir, Sanjiv S; Cheng, Zhen; Robbins, Robert C; Beygui, Ramin E

    2011-09-01

    Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.

  2. Designing a Binding Interface for Control of Cancer Cell Adhesion via 3D Topography and Metabolic Oligosaccharide Engineering

    PubMed Central

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

    2011-01-01

    This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac5ManNTGc, a thiol-bearing analogue of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bioorthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion. PMID:21549424

  3. Improved structural quality of AlN grown on sapphire by 3D/2D alternation growth

    NASA Astrophysics Data System (ADS)

    Guo, Yanmin; Fang, Yulong; Yin, Jiayun; Zhang, Zhirong; Wang, Bo; Li, Jia; Lu, Weili; Feng, Zhihong

    2017-04-01

    Three dimensional (3D) and two dimensional (2D) alternation growth was used to grow AlN epitaxial layers on sapphire substrates. AlN samples grown using this technique have higher crystalline quality and lower dislocation density than samples grown using only 3D or 2D growth modes as witnessed by the high-resolution X-ray diffraction. Smooth atomic terraces with root mean square roughness of 0.107 nm were observed using atomic force microscopy (AFM) when the 3D and 2D AlN were 75 nm and 425 nm, respectively. This sample possesses single crystallographic orientation along the c-axis identified by Raman spectroscopy. Furthermore, the 3D/2D alternating growth mode modulates internal stress in AlN epitaxial layer by adjusting 2D AlN thickness, and the mechanism was studied in detail.

  4. Bridging the gap: from 2D cell culture to 3D microengineered extracellular matrices

    PubMed Central

    Li, Yanfen

    2016-01-01

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

  5. 3D Cell Printing of Functional Skeletal Muscle Constructs Using Skeletal Muscle-Derived Bioink.

    PubMed

    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.

  6. 3D Models of the NCI60 Cell Lines for Screening Oncology Compounds.

    PubMed

    Selby, Mike; Delosh, Rene; Laudeman, Julie; Ogle, Chad; Reinhart, Russell; Silvers, Thomas; Lawrence, Scott; Kinders, Robert; Parchment, Ralph; Teicher, Beverly A; Evans, David M

    2017-03-01

    The NCI60 cell line panel screen includes 60 human tumor cell lines derived from nine tumor types that has been used over the past 20+ years to screen small molecules, biologics, and natural products for activity. Cells in monolayer culture in 96-well plates are exposed to compounds for 48 h, and Sulforhodamine B is used to determine cell viability. Data analysis tools such as COMPARE allow classification of compounds based on the pattern of cell line response. However, many compounds highly active in monolayer cell culture fail to show efficacy in vivo. Therefore, we explored 3D culture of the NCI60 panel as a strategy to improve the predictive accuracy of the screen. 3D cultures more closely resemble tumors than monolayer cultures with tighter cell-cell contact and nutrient and oxygen gradients between the periphery and the center. We optimized the NCI60 cell line panel for generating 3D spheroids of a prespecified diameter (300-500 µm) in ultra-low attachment (ULA) plates. Spheroids were classified into four categories based on imaging, and concentration response of select agents in 2D and 3D models is presented.

  7. Paper/PMMA Hybrid 3D Cell Culture Microfluidic Platform for the Study of Cellular Crosstalk.

    PubMed

    Lei, Kin Fong; Chang, Chih-Hsuan; Chen, Ming-Jie

    2017-04-06

    Studying cellular crosstalk is important for understanding tumor initiation, progression, metastasis, and therapeutic resistance. Moreover, a three-dimensional (3D) cell culture model can provide a more physiologically meaningful culture microenvironment. However, studying cellular crosstalk in a 3D cell culture model involves tedious processing. In this study, a paper/poly(methyl methacrylate) (PMMA) hybrid 3D cell culture microfluidic platform was successfully developed for the study of cellular crosstalk. The platform was a paper substrate with culture microreactors placed on a PMMA substrate with hydrogel-infused channels. Different types of cells were directly seeded and cultured in the microreactors. Aberrant cell proliferation of the affected cells was induced by secretions from transfected cells, and the proliferation ratios were investigated using a colorimetric method. The results showed that the responses of cellular crosstalk were different in different types of cells. Moreover, neutralizing and competitive assays were performed to show the functionality of the platform. Additionally, the triggered signaling pathways of the affected cells were directly analyzed by a subsequent immunoassay. The microfluidic platform provides a simple method for studying cellular crosstalk and the corresponding signaling pathways in a 3D culture model.

  8. 3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:25482337

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

    PubMed

    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.

  12. Mechanosensing of cells in 3D gel matrices based on natural and synthetic materials.

    PubMed

    Shan, Jieling; Chi, Qingjia; Wang, Hongbing; Huang, Qiping; Yang, Li; Yu, Guanglei; Zou, Xiaobing

    2014-11-01

    Cells in vivo typically are found in 3D matrices, the mechanical stiffness of which is important to the cell and tissue-scale biological processes. Although it is well characterized that as to how cells sense matrix stiffness in 2D substrates, the scenario in 3D matrices needs to be explored. Thus, materials that can mimic native 3D environments and possess wide, physiologically relevant elasticity are highly desirable. Natural polymer-based materials and synthetic hydrogels could provide an better 3D platforms to investigate the mechano-response of cells with stiffness comparable to their native environments. However, the limited stiffness range together with interdependence of matrix stiffness and adhesive ligand density are inherent in many kinds of materials, and hinder efforts to demonstrate the true effects contributed by matrix stiffness. These problems have been addressed by the recently emerging exquisitely designed materials based on native matrix components, designer matrices, and synthetic polymers. In this review, a variety of materials with a wide stiffness range that mimic the mechanical environment of native 3D matrices and the independent affection of stiffness for cellular behavior and tissue-level processes are discussed.

  13. Characterisation of the surface structure of 3D printed scaffolds for cell infiltration and surgical suturing.

    PubMed

    Ruiz-Cantu, Laura; Gleadall, Andrew; Faris, Callum; Segal, Joel; Shakesheff, Kevin; Yang, Jing

    2016-03-01

    3D printing is of great interest for tissue engineering scaffolds due to the ability to form complex geometries and control internal structures, including porosity and pore size. The porous structure of scaffolds plays an important role in cell ingrowth and nutrition infusion. Although the internal porosity and pore size of 3D printed scaffolds have been frequently studied, the surface porosity and pore size, which are critical for cell infiltration and mass transport, have not been investigated. The surface geometry can differ considerably from the internal scaffold structure depending on the 3D printing process. It is vital to be able to control the surface geometry of scaffolds as well as the internal structure to fabricate optimal architectures. This work presents a method to control the surface porosity and pore size of 3D printed scaffolds. Six scaffold designs have been printed with surface porosities ranging from 3% to 21%. We have characterised the overall scaffold porosity and surface porosity using optical microscopy and microCT. It has been found that surface porosity has a significant impact on cell infiltration and proliferation. In addition, the porosity of the surface has been found to have an effect on mechanical properties and on the forces required to penetrate the scaffold with a surgical suturing needle. To the authors' knowledge, this study is the first to investigate the surface geometry of extrusion-based 3D printed scaffolds and demonstrates the importance of surface geometry in cell infiltration and clinical manipulation.

  14. Direct cell writing of 3D microorgan for in vitro pharmacokinetic model.

    PubMed

    Chang, Robert; Nam, Jae; Sun, Wei

    2008-06-01

    A novel targeted application of tissue engineering is the development of an in vitro pharmacokinetic model for drug screening and toxicology. An in vitro pharmacokinetic model is needed to realistically and reliably predict in vivo human response to drug administrations and potential toxic exposures. This paper details the fabrication process development and adaptation of microfluidic devices for the creation of such a physiologically relevant pharmacokinetic model. First, an automated syringe-based, layered direct cell writing (DCW) bioprinting process creates a 3D microorgan that biomimics the cell's natural microenvironment with enhanced functionality. Next, soft lithographic micropatterning techniques are used to fabricate a microscale in vitro device to house the 3D microorgan. This paper demonstrates the feasibility of the DCW process for freeform biofabrication of 3D cell-encapsulated hydrogel-based tissue constructs with defined reproducible patterns, direct integration of 3D constructs onto a microfluidic device for continuous perfusion drug flow, and characterization of 3D tissue constructs with predictable cell viability/proliferation outcomes and enhanced functionality over traditional culture methods.

  15. Altering the Microenvironment to Promote Dormancy of Metastatic Breast Cancer Cell in a 3D Bone Culture System

    DTIC Science & Technology

    2014-04-01

    for metastatic breast cancer cells to grow or remain dormant. This hypothesis is being tested using a 3D bioreactor of ECM, derived from osteoblasts...dormant human cells to proliferate in the bioreactor in co-culture with OB. The effect appears to depend on prostaglandin production. Chronic...growth of cancer cells, murine osteoblasts, MC3T3-E1, were grown for 2 months in the bioreactor with a basal medium of αMEM with 10mM β

  16. 3-D confocal laser scanning microscopy used in morphometric analysis of rat Purkinje cell dendritic spines after chronic ethanol consumption.

    PubMed

    Wenisch, S; Fortmann, B; Steinmetz, T; Kriete, A; Leiser, R; Bitsch, I

    1998-12-01

    A confocal laser scanning microscope (with a 543 nm laser) was used for imaging rat Purkinje cell dendritic spines at high 3-D resolution. In a nutritionally controlled study of the rat, 5 months of ethanol consumption was demonstrated to alter the spines of Purkinje cell dendrites in rat cerebellum. Intact spines showed significant elongation after ethanol exposure, whereas this neuromorphological alteration could not be detected in controls. Spine elongation could be regarded as compensative growth of spines in search of new synaptic contacts due to alcohol induced cell loss.

  17. Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber.

    PubMed

    Haessler, Ulrike; Teo, Jeremy C M; Foretay, Didier; Renaud, Philippe; Swartz, Melody A

    2012-04-01

    The migration of cells such as leukocytes, tumor cells, and fibroblasts through 3D matrices is critical for regulating homeostasis and immunity and for driving pathogenesis. Interstitial flow through the extracellular matrix, which can substantially increase during inflammation and in the tumor microenvironment, can influence cell migration in multiple ways. Leukocytes and tumor cells are heterogeneous in their migration responses to flow, yet most 3D migration studies use endpoint measurements representing average characteristics. Here we present a robust new microfluidic device for 3D culture with live imaging under well-controlled flow conditions, along with a comparison of analytical methods for describing the migration behavior of heterogeneous cell populations. We then use the model to provide new insight on how interstitial flow affects MDA-MB-231 breast cancer cell invasion, phenomena that are not seen from averaged or endpoint measurements. Specifically, we find that interstitial flow increases the percentage of cells that become migratory, and increases migrational speed in about 20% of the cells. It also increases the migrational persistence of a subpopulation (5-10% of cells) in the positive or negative flow direction. Cells that migrated upstream moved faster but with less directedness, whereas cells that migrated in the direction of flow moved at slower speeds but with higher directedness. These findings demonstrate how fluid flow in the tumor microenvironment can enhance tumor cell invasion by directing a subpopulation of tumor cells in the flow direction; i.e., towards the draining lymphatic vessels, a major route of metastasis.

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

    SciTech Connect

    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. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  19. Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells

    PubMed Central

    Robinson, Benjamin K.; Cortes, Ernesto; Rice, Alistair J.; Sarper, Muge

    2016-01-01

    ABSTRACT Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer. Until recently, most cellular studies have been conducted on 2D environments where mechanical cues significantly differ from physiologically relevant 3D environments, impacting cellular behaviour and masking the interpretation of cellular function in health and disease. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling. Monitoring and quantification of collagen-I structure in remodelled matrices, through designated algorithms, show that 3D matrices can be used to correlate remodelling with increased ECM stiffness observed in fibrosis. Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment. PMID:27170254

  20. Nanoscale Analysis of a Hierarchical Hybrid Solar Cell in 3D

    PubMed Central

    Divitini, Giorgio; Stenzel, Ole; Ghadirzadeh, Ali; Guarnera, Simone; Russo, Valeria; Casari, Carlo S; Bassi, Andrea Li; Petrozza, Annamaria; Di Fonzo, Fabio; Schmidt, Volker; Ducati, Caterina

    2014-01-01

    A quantitative method for the characterization of nanoscale 3D morphology is applied to the investigation of a hybrid solar cell based on a novel hierarchical nanostructured photoanode. A cross section of the solar cell device is prepared by focused ion beam milling in a micropillar geometry, which allows a detailed 3D reconstruction of the titania photoanode by electron tomography. It is found that the hierarchical titania nanostructure facilitates polymer infiltration, thus favoring intermixing of the two semiconducting phases, essential for charge separation. The 3D nanoparticle network is analyzed with tools from stochastic geometry to extract information related to the charge transport in the hierarchical solar cell. In particular, the experimental dataset allows direct visualization of the percolation pathways that contribute to the photocurrent. PMID:25834481

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

    PubMed

    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.

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

    PubMed Central

    Joshi, Pranav; Lee, Moo-Yeal

    2015-01-01

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

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2016-07-01

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

  5. Ultrafine fibrous gelatin scaffolds with deep cell infiltration mimicking 3D ECMs for soft tissue repair.

    PubMed

    Jiang, Qiuran; Xu, Helan; Cai, Shaobo; Yang, Yiqi

    2014-07-01

    In this research, ultrafine fibrous scaffolds with deep cell infiltration and sufficient water stability have been developed from gelatin, aiming to mimic the extracellular matrices (ECMs) as three dimensional (3D) stromas for soft tissue repair. The ultrafine fibrous scaffolds produced from the current technologies of electrospinning and phase separation are either lack of 3D oriented fibrous structure or too compact to be penetrated by cells. Whilst electrospun scaffolds are able to emulate two dimensional (2D) ECMs, they cannot mimic the 3D ECM stroma. In this work, ultralow concentration phase separation (ULCPS) has been developed to fabricate gelatin scaffolds with 3D randomly oriented ultrafine fibers and loose structures. Besides, a non-toxic citric acid crosslinking system has been established for the ULCPS method. This system could endow the scaffolds with sufficient water stability, while maintain the fibrous structures of scaffolds. Comparing with electrospun scaffolds, the ULCPS scaffolds showed improved cytocompatibility and more importantly, cell infiltration. This research has proved the possibility of using gelatin ULCPS scaffolds as the substitutes of 3D ECMs.

  6. On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology

    PubMed Central

    Shao, Yue; Sang, Jianming; Fu, Jianping

    2015-01-01

    Human pluripotent stem cells (hPSCs) provide promising resources for regenerating tissues and organs and modeling development and diseases in vitro. To fulfill their promise, the fate, function, and organization of hPSCs need to be precisely regulated in a three-dimensional (3D) environment to mimic cellular structures and functions of native tissues and organs. In the past decade, innovations in 3D culture systems with functional biomaterials have enabled efficient and versatile control of hPSC fate at the cellular level. However, we are just at the beginning of bringing hPSC-based regeneration and development and disease modeling to the tissue and organ levels. In this review, we summarize existing bioengineered culture platforms for controlling hPSC fate and function by regulating inductive mechanical and biochemical cues coexisting in the synthetic cell microenvironment. We highlight recent excitements in developing 3D hPSC-based in vitro tissue and organ models with in vivo-like cellular structures, interactions, and functions. We further discuss an emerging multifaceted mechanotransductive signaling network – with transcriptional coactivators YAP and TAZ at the center stage – that regulate fates and behaviors of mammalian cells, including hPSCs. Future development of 3D biomaterial systems should incorporate dynamically modulated mechanical and chemical properties targeting specific intracellular signaling events leading to desirable hPSC fate patterning and functional tissue formation in 3D. PMID:25818411

  7. Single cell detection using 3D magnetic rolled-up structures.

    PubMed

    Ger, Tzong-Rong; Huang, Hao-Ting; Huang, Chen-Yu; Lai, Mei-Feng

    2013-11-07

    A 3D rolled-up structure made of a SiO2 layer and a fishbone-like magnetic thin film was proposed here as a biosensor. The magnetoresistance (MR) measurement results of the sensor suggest that the presence of the stray field, which is induced by the magnetic nanoparticles, significantly increased the switching field. Comparing the performance of the 2D sensor and 3D sensor designed in this study, the response in switching field variation was 12.14% in the 2D sensor and 62.55% in the 3D sensor. The response in MR ratio variation was 4.55% in the 2D sensor and 82.32% in the 3D sensor. In addition, the design of the 3D sensor structure also helped to attract and trap a single magnetic cell due to its stronger stray field compared with the 2D structure. The 3D magnetic biosensor designed here can provide important information for future biochip research and applications.

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

    PubMed

    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.

  9. Activating the nuclear piston mechanism of 3D migration in tumor cells.

    PubMed

    Petrie, Ryan J; Harlin, Heather M; Korsak, Lulu I T; Yamada, Kenneth M

    2017-01-02

    Primary human fibroblasts have the remarkable ability to use their nucleus like a piston, switching from low- to high-pressure protrusions in response to the surrounding three-dimensional (3D) matrix. Although migrating tumor cells can also change how they migrate in response to the 3D matrix, it is not clear if they can switch between high- and low-pressure protrusions like primary fibroblasts. We report that unlike primary fibroblasts, the nuclear piston is not active in fibrosarcoma cells. Protease inhibition rescued the nuclear piston mechanism in polarized HT1080 and SW684 cells and generated compartmentalized pressure. Achieving compartmentalized pressure required the nucleoskeleton-cytoskeleton linker protein nesprin 3, actomyosin contractility, and integrin-mediated adhesion, consistent with lobopodia-based fibroblast migration. In addition, this activation of the nuclear piston mechanism slowed the 3D movement of HT1080 cells. Together, these data indicate that inhibiting protease activity during polarized tumor cell 3D migration is sufficient to restore the nuclear piston migration mechanism with compartmentalized pressure characteristic of nonmalignant cells.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    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.

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

    PubMed Central

    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 that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3-D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme and cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations. PMID:23447378

  13. 3D Silicon Microstructures: A New Tool for Evaluating Biological Aggressiveness of Tumor Cells.

    PubMed

    Mazzini, Giuliano; Carpignano, Francesca; Surdo, Salvatore; Aredia, Francesca; Panini, Nicolò; Torchio, Martina; Erba, Eugenio; Danova, Marco; Scovassi, Anna Ivana; Barillaro, Giuseppe; Merlo, Sabina

    2015-10-01

    In this work, silicon micromachined structures (SMS), consisting of arrays of 3- μ m-thick silicon walls separated by 50- μm-deep, 5- μ m-wide gaps, were applied to investigate the behavior of eight tumor cell lines, with different origins and biological aggressiveness, in a three-dimensional (3D) microenvironment. Several cell culture experiments were performed on 3D-SMS and cells grown on silicon were stained for fluorescence microscopy analyses. Most of the tumor cell lines recognized in the literature as highly aggressive (OVCAR-5, A375, MDA-MB-231, and RPMI-7951) exhibited a great ability to enter and colonize the narrow deep gaps of the SMS, whereas less aggressive cell lines (OVCAR-3, Capan-1, MCF7, and NCI-H2126) demonstrated less penetration capability and tended to remain on top of the SMS. Quantitative image analyses of several fluorescence microscopy fields of silicon samples were performed for automatic cell recognition and count, in order to quantify the fraction of cells inside the gaps, with respect to the total number of cells in the examined field. Our results show that higher fractions of cells in the gaps are obtained with more aggressive cell lines, thus supporting in a quantitative way the observation that the behavior of tumor cells on the 3D-SMS depends on their aggressiveness level.

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

    PubMed

    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

    2015-04-02

    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.

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

    PubMed Central

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

    2014-01-01

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

  16. Architectural proteins: regulators of 3D genome organization in cell fate.

    PubMed

    Gómez-Díaz, Elena; Corces, Victor G

    2014-11-01

    The relation between alterations in chromatin structure and changes in gene expression during cell differentiation has served as a paradigm to understand the link between genome organization and function. Yet, the factors involved and the mechanisms by which the 3D organization of the nucleus is established remain poorly understood. The use of Chromosome Conformation-Capture (3C)-based approaches has resulted in a new appreciation of the role of architectural proteins in the establishment of 3D genome organization. Architectural proteins orchestrate higher-order chromatin organization through the establishment of interactions between regulatory elements across multiple spatial scales. The regulation of these proteins, their interaction with DNA, and their co-occurrence in the genome, may be responsible for the plasticity of 3D chromatin architecture that dictates cell and time-specific blueprints of gene expression.

  17. Mesoscopic in vivo 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography.

    PubMed

    Chen, Lingling; Alexandrov, Yuriy; Kumar, Sunil; Andrews, Natalie; Dallman, Margaret J; French, Paul M W; McGinty, James

    2015-04-01

    We describe an angular multiplexed imaging technique for 3-D in vivo cell tracking of sparse cell distributions and optical projection tomography (OPT) with superior time-lapse resolution and a significantly reduced light dose compared to volumetric time-lapse techniques. We demonstrate that using dual axis OPT, where two images are acquired simultaneously at different projection angles, can enable localization and tracking of features in 3-D with a time resolution equal to the camera frame rate. This is achieved with a 200x reduction in light dose compared to an equivalent volumetric time-lapse single camera OPT acquisition with 200 projection angles. We demonstrate the application of this technique to mapping the 3-D neutrophil migration pattern observed over ~25.5 minutes in a live 2 day post-fertilisation transgenic LysC:GFP zebrafish embryo following a tail wound.

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

    PubMed

    Borau, C; Kamm, R D; García-Aznar, J M

    2011-12-01

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

  19. Novel 3-D cell culture system for in vitro evaluation of anticancer drugs under anchorage-independent conditions.

    PubMed

    Aihara, Ayako; Abe, Natsuki; Saruhashi, Koichiro; Kanaki, Tatsuro; Nishino, Taito

    2016-12-01

    Anticancer drug discovery efforts have used 2-D cell-based assay models, which fail to forecast in vivo efficacy and result in a lower success rate of clinical approval. Recent 3-D cell culture models are expected to bridge the gap between 2-D and in vivo models. However, 3-D cell culture methods that are available for practical anticancer drug screening have not yet been fully attained. In this study, we screened several polymers for their ability to suspend cells or cell spheroids homogeneously in a liquid medium without changing the viscosity behavior, and identified gellan gum (FP001), as the most potent polymer. FP001 promoted cell dispersion in the medium and improved the proliferation of a wide range of cancer cell lines under low attachment conditions by inhibiting the formation of large-sized spheroids. In addition, cancer cells cultured with FP001-containing medium were more susceptible to inhibitors of epidermal growth factor (EGF) signaling than those cultured under attachment conditions. We also showed that ligands of the EGF receptor family clearly enhance proliferation of SKOV3 ovarian carcinoma cells under anchorage-independent conditions with FP001. Consistent with this result, the cells grown with FP001 showed higher EGF receptor content compared with cells cultured under attachment conditions. In conclusion, we developed a novel 3-D cell culture system that is available for high throughput screening of anticancer agents, and is suitable for evaluation of molecular-targeted anticancer drugs. Three-dimensional cell culture using FP001 will be of value in the development of useful technologies for anticancer drug discovery.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  3. Bulge Growth and Quenching Since Z=2.5 in Candels/3D-HST

    NASA Technical Reports Server (NTRS)

    Lang, Phillip; Wuyts, Stijn; Somerville, Rachel S.; Schreiber, Natascha M. Foerster; Genzel, Reinhard; Bell, Eric F.; Brammer, Gabe; Dekel, Avishai; Faber, Sandra M.; Ferguson, Henry C.; Grogin, Norman A.; Kocevski, Dale D.; Koekemoer, Anton M.; Lutz, Dieter; McGrath, Elizabeth J.; Momcheva, Ivelina; Nelson, Erica J.; Primack, Joel R.; Rosario, David J.; Skelton, Rosalind E.; Tacconi, Linda J.; van Dokkum, Peter G.; Whitaker, Katherine E.

    2014-01-01

    Exploiting the deep high-resolution imaging of all 5 CANDELS fields, and accurate redshift informationprovided by 3D-HST, we investigate the relation between structure and stellar populations fora mass-selected sample of 6764 galaxies above 1010 M, spanning the redshift range 0.5 z 2.5.For the first time, we fit 2-dimensional models comprising a single Sersic fit and two-component (i.e.,bulge + disk) decompositions not only to the H-band light distributions, but also to the stellar massmaps reconstructed from resolved stellar population modeling. We confirm that the increased bulgeprominence among quiescent galaxies, as reported previously based on rest-optical observations, remainsin place when considering the distributions of stellar mass. Moreover, we observe an increaseof the typical Sersic index and bulge-to-total ratio (with median BT reaching 40-50) among starforminggalaxies above 1011 M. Given that quenching for these most massive systems is likely tobe imminent, our findings suggest that significant bulge growth precedes a departure from the starformingmain sequence. We demonstrate that the bulge mass (and ideally knowledge of the bulge andtotal mass) is a more reliable predictor of the star-forming versus quiescent state of a galaxy thanthe total stellar mass. The same trends are predicted by the state-of-the-art semi-analytic model bySomerville et al. In the latter, bulges and black holes grow hand in hand through merging andordisk instabilities, and AGN-feedback shuts off star formation. Further observations will be requiredto pin down star formation quenching mechanisms, but our results imply they must be internal to thegalaxies and closely associated with bulge growth.

  4. Bulge growth and quenching since z = 2.5 in CANDELS/3D-HST

    SciTech Connect

    Lang, Philipp; Wuyts, Stijn; Schreiber, Natascha M. Förster; Genzel, Reinhard; Lutz, Dieter; Rosario, David J.; Somerville, Rachel S.; Bell, Eric F.; Brammer, Gabe; Dekel, Avishai; Faber, Sandra M.; Momcheva, Ivelina; Kocevski, Dale D.; McGrath, Elizabeth J.; Nelson, Erica J.; Primack, Joel R.; Skelton, Rosalind E.; and others

    2014-06-10

    Exploiting the deep high-resolution imaging of all five CANDELS fields, and accurate redshift information provided by 3D-HST, we investigate the relation between structure and stellar populations for a mass-selected sample of 6764 galaxies above 10{sup 10} M {sub ☉}, spanning the redshift range 0.5 < z < 2.5. For the first time, we fit two-dimensional models comprising a single Sérsic fit and two-component (i.e., bulge + disk) decompositions not only to the H-band light distributions, but also to the stellar mass maps reconstructed from resolved stellar population modeling. We confirm that the increased bulge prominence among quiescent galaxies, as reported previously based on rest-optical observations, remains in place when considering the distributions of stellar mass. Moreover, we observe an increase of the typical Sérsic index and bulge-to-total ratio (with median B/T reaching 40%-50%) among star-forming galaxies above 10{sup 11} M {sub ☉}. Given that quenching for these most massive systems is likely to be imminent, our findings suggest that significant bulge growth precedes a departure from the star-forming main sequence. We demonstrate that the bulge mass (and ideally knowledge of the bulge and total mass) is a more reliable predictor of the star-forming versus quiescent state of a galaxy than the total stellar mass. The same trends are predicted by the state-of-the-art, semi-analytic model by Somerville et al. In this model, bulges and black holes grow hand in hand through merging and/or disk instabilities, and feedback from active galactic nuclei shuts off star formation. Further observations will be required to pin down star formation quenching mechanisms, but our results imply that they must be internal to the galaxies and closely associated with bulge growth.

  5. Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D

    PubMed Central

    Yang, Fan; Cho, Seung-Woo; Son, Sun Mi; Hudson, Sarah P.; Bogatyrev, Said; Keung, Lily; Kohane, Daniel S.; Langer, Robert

    2010-01-01

    Embryonic stem cells (ESCs) are promising cell sources for tissue engineering and regenerative medicine. Scaffolds for ESC-based tissue regeneration should provide not only structural support, but also signals capable of supporting appropriate cell differentiation and tissue development. Extracellular matrix (ECM) is a key component of stem cell niche in vivo and can influence stem cell fate via mediating cell attachment and migration, presenting chemical and physical cues, as well as binding soluble factors. Here we investigated the effects of combinatorial extracellular matrix proteins on controlled human ESC (hESC) differentiation. Varying ECM compositions in 3D markedly affects cell behavior, and optimal compositions of ECM hydrogels are identified which facilitate specific-lineage differentiation of stem cells. To our knowledge, this is the first combinatorial analysis of ECM hydrogels for their effects on hESC differentiation in 3D. The 3D matrices described herein may provide a useful platform for studying the interactive ECM signaling in influencing stem cell differentiation. PMID:20614932

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

    PubMed

    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.

  7. Dextran-based hydrogel formed by thiol-Michael addition reaction for 3D cell encapsulation.

    PubMed

    Liu, Zhen Qi; Wei, Zhao; Zhu, Xv Long; Huang, Guo You; Xu, Feng; Yang, Jian Hai; Osada, Yoshihito; Zrínyi, Miklós; Li, Jian Hui; Chen, Yong Mei

    2015-04-01

    Cell encapsulation in three-dimensional (3D) hydrogels can mimic native cell microenvironment and plays a major role in cell-based transplantation therapies. In this contribution, a novel in situ-forming hydrogel, Dex-l-DTT hydrogel ("l" means "linked-by"), by cross-linking glycidyl methacrylate derivatized dextran (Dex-GMA) and dithiothreitol (DTT) under physiological conditions, has been developed using thiol-Michael addition reaction. The mechanical properties, gelation process and degree of swelling of the hydrogel can be easily adjusted by changing the pH of phosphate buffer saline. The 3D cell encapsulation ability is demonstrated by encapsulating rat bone marrow mesenchymal stem cells (BMSCs) and NIH/3T3 fibroblasts into the in situ-forming hydrogel with maintained high viability. The BMSCs also maintain their differentiation potential after encapsulation. These results demonstrate that the Dex-l-DTT hydrogel holds great potential for biomedical field.

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

    PubMed Central

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

    2014-01-01

    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 105 hMSCs realized better ischemic limb salvage than treatment with 106 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. PMID:25197069

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

    PubMed Central

    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

  10. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds.

    PubMed

    Lee, Wonjae; Park, Jon

    2016-07-06

    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.

  11. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    PubMed Central

    Lee, Wonjae; Park, Jon

    2016-01-01

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

  12. 3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds

    NASA Astrophysics Data System (ADS)

    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.

  13. Microscale technologies for imaging endogenous gene expression in individual cells within 3D tissues

    NASA Astrophysics Data System (ADS)

    Ye, Ting; Luo, Zhen; Ma, Yunzhe; Gill, Harvinder Singh; Nitin, N.

    2013-05-01

    The goal of this study was to develop an innovative approach to image gene expression in intact 3D tissues. Imaging gene expression of individual cells in 3D tissues is expected to have a significant impact on both clinical diagnostic applications and fundamental biological science and engineering applications in a laboratory setting. To achieve this goal, we have developed an integrated approach that combines: 1) microneedle-based minimally invasive intra-tissue delivery of oligonucleotide probes and Streptolysin O (SLO) or CPP; 2) SLO as a pore forming permeation enhancer to enable intracellular delivery of oligonucleotide probes and CPP peptides can also transport conjugated cargo in cells; and 3) fluorescence resonance energy transfer (FRET) pair of ON probes to improve specificity and sensitivity of RNA detection in tissue models. The results of this study demonstrate uniform coating and rapid release of ON probes from microneedles in a tissue environment. Microneedle assisted delivery of ON probes in 3D tissue does not result in cell damage and the ON probes are uniformly delivered in the tissue. The results also demonstrate the feasibility of FRET imaging of ON probes in 3D tissue and highlight the potential for imaging 28-s rRNA in individual living cells.

  14. 3D Cultures of prostate cancer cells cultured in a novel high-throughput culture platform are more resistant to chemotherapeutics compared to cells cultured in monolayer.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    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

  16. Optimizing Photo-Encapsulation Viability of Heart Valve Cell Types in 3D Printable Composite Hydrogels.

    PubMed

    Kang, Laura Hockaday; Armstrong, Patrick A; Lee, Lauren Julia; Duan, Bin; Kang, Kevin Heeyong; Butcher, Jonathan Talbot

    2017-02-01

    Photocrosslinking hydrogel technologies are attractive for the biofabrication of cardiovascular soft tissues, but 3D printing success is dependent on multiple variables. In this study we systematically test variables associated with photocrosslinking hydrogels (photoinitiator type, photoinitiator concentration, and light intensity) for their effects on encapsulated cells in an extrusion 3D printable mixture of methacrylated gelatin/poly-ethylene glycol diacrylate/alginate (MEGEL/PEGDA3350/alginate). The fabrication conditions that produced desired hydrogel mechanical properties were compared against those that optimize aortic valve or mesenchymal stem cell viability. In the 3D hydrogel culture environment and fabrication setting studied, Irgacure can increase hydrogel stiffness with a lower proportional decrease in encapsulated cell viability compared to VA086. Human adipose derived mesenchymal stem cells (HADMSC) survived increasing photoinitiator concentrations in photo-encapsulation conditions better than aortic valve interstitial cells (HAVIC) and aortic valve sinus smooth muscle cells (HASSMC). Within the range of photo-encapsulation fabrication conditions tested with MEGEL/PEGDA/alginate (0.25-1.0% w/v VA086, 0.025-0.1% w/v Irgacure 2959, and 365 nm light intensity 2-136 mW/cm(2)), the highest viabilities achieved were 95, 93, and 93% live for HASSMC, HAVIC, and HADMSC respectively. These results identify parameter combinations that optimize cell viability during 3D printing for multiple cell types. These results also indicate that general oxidative stress is higher in photocrosslinking conditions that induce lower cell viability. However, suppressing this increase in intracellular oxidative stress did not improve cell viability, which suggests that other stress mechanisms also contribute.

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

    PubMed Central

    Fai, Stephen; Bennett, Steffany A.L.

    2010-01-01

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

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

    PubMed

    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.

  19. A novel 3D integrated platform for the high-resolution study of cell migration plasticity.

    PubMed

    Schneider, Julian; Bachmann, Tobias; Franco, Davide; Richner, Patrizia; Galliker, Patrick; Tiwari, Manish K; Ferrari, Aldo; Poulikakos, Dimos

    2013-08-01

    Understanding the mechanisms of interstitial cancer migration is of great scientific and medical interest. Creating 3D platforms, conducive to optical microscopy and mimicking the physical parameters (in plane and out of plane) involved in interstitial migration, is a major step forward in this direction. Here, a novel approach is used to directly print free-form, 3D micropores on basal scaffolds containing microgratings optimized for contact guidance. The platforms so formed are validated by monitoring cancer cell migration and micropore penetration with high-resolution optical microscopy. The shapes, sizes and deformability of the micropores are controllable, paving the way to decipher their role in interstitial migration.

  20. 3-D measurement of osmotic dehydration of isolated and adhered PC-3 cells.

    PubMed

    Yoshimori, Takashi; Takamatsu, Hiroshi

    2009-02-01

    Cell dehydration during freezing results from an elevated concentration of electrolytes in the extracellular medium that is deeply involved in cellular injury. We undertook real-time threedimensional (3-D) observation of osmotic dehydration of cells, motivated by a comparison of cellular responses between isolated cells in suspension and cultured cells adhering to a surface since several studies have suggested a difference in freeze tolerance between cell suspensions and monolayers. A laser confocal scanner was used with a perfusion microscope to capture sectional images of chloromethylbenzamido (DiI)-stained PC-3 cells that were exposed to an increase in NaCl concentration from 0.15 to 0.5M at 23 degrees C. Change in cell volume was determined from reconstructed 3-D images taken every 2.5s. When cells were exposed to an elevated NaCl concentration, isolated cells contracted and markedly distorted from their original spherical shape. In contrast, adhered cells showed only a reduction in height and kept their basal area constant. Apparent membrane hydraulic conductivity did not vary considerably between isolated and adhered cells, suggesting a negligible effect of the cytoskeletal structure on the rate of water transport. The surface area that contributed to water transport in adhered PC-3 cells was nearly equal to or slightly smaller than that present in isolated cells. Therefore, the similarity in properties and dimensions between isolated and adhered cells indicate that there will be similar extents of dehydration, resulting in a similar degree of supercooling during freezing.

  1. Three-dimensional (3D) hydrodynamic focusing for continuous sampling and analysis of adherent cells.

    PubMed

    Xu, Chunxiu; Wang, Min; Yin, Xuefeng

    2011-10-07

    A simple three-dimensional (3D) hydrodynamic focusing microfluidic device integrated with continuous sampling, rapid dynamic lysis, capillary electrophoretic (CE) separation and detection of intracellular content is presented. One of the major difficulties in microfluidic cell analysis for adherent cells is that the cells are prone to attaching to the channel surface. To solve this problem, a cross microfluidic chip with three sheath-flow channels located on both sides of and below the sampling channel was developed. With the three sheath flows around the sample solution-containing cells, the formed soft fluid wall prevents the cells from adhering to the channel surface. Labeled cells were 3D hydrodynamically focused by the sheath-flow streams and smoothly introduced into the cross-section one by one. The introduction of sheath-flow streams not only ensured single-cell sampling but avoided blockage of the sampling channel by adherent cells as well. The maximum rate for introduction of individual cells into the separation channel was about 151 cells min(-1). With electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 400 ms at the entry of the channel by sodium dodecylsulfate (SDS) added in the sheath-flow solution. The microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single HepG2 cells. The average analysis throughput of ROS and GSH in single cells was 16-18 cells min(-1).

  2. Visualization and 3D Reconstruction of Flame Cells of Taenia solium (Cestoda)

    PubMed Central

    Valverde-Islas, Laura E.; Arrangoiz, Esteban; Vega, Elio; Robert, Lilia; Villanueva, Rafael; Reynoso-Ducoing, Olivia; Willms, Kaethe; Zepeda-Rodríguez, Armando; Fortoul, Teresa I.; Ambrosio, Javier R.

    2011-01-01

    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. PMID:21412407

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Investigation on 3D morphological changes of in vitro cells through digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Memmolo, Pasquale; Miccio, Lisa; Merola, Francesco; Netti, Paolo A.; Coppola, Giuseppe; Ferraro, Pietro

    2013-04-01

    We report the investigation of the identification and measurement of region of interest (ROI) in quantitative phase-contrast maps (QPMs) of biological cells by digital holographic microscopy (DHM), with the aim to analyze the 3D positions and 3D morphology together. We consider as test case for our tool the in vitro bull sperm head morphometry analysis. Extraction and measurement of various morphological parameters are performed by using two methods: the anisotropic diffusion filter, that is based on the Gaussian diffusivity function which allows more accuracy of the edge position, and the simple thresholding filter. In particular we consider the calculation of area, ellipticity, perimeter, major axis, minor axis and shape factor as a morphological parameter, instead, for the estimation of 3D position, we compute the centroid, the weighted centroid and the maximum phase values. A statistical analysis on a data set composed by N = 14 holograms relative to bovine spermatozoa and its reference holograms is reported.

  5. 3D niche microarrays for systems-level analyses of cell fate.

    PubMed

    Ranga, A; Gobaa, S; Okawa, Y; Mosiewicz, K; Negro, A; Lutolf, M P

    2014-07-14

    The behaviour of mammalian cells in a tissue is governed by the three-dimensional (3D) microenvironment and involves a dynamic interplay between biochemical and mechanical signals provided by the extracellular matrix (ECM), cell-cell interactions and soluble factors. The complexity of the microenvironment and the context-dependent cell responses that arise from these interactions have posed a major challenge to understanding the underlying regulatory mechanisms. Here we develop an experimental paradigm to dissect the role of various interacting factors by simultaneously synthesizing more than 1,000 unique microenvironments with robotic nanolitre liquid-dispensing technology and by probing their effects on cell fate. Using this novel 3D microarray platform, we assess the combined effects of matrix elasticity, proteolytic degradability and three distinct classes of signalling proteins on mouse embryonic stem cells, unveiling a comprehensive map of interactions involved in regulating self-renewal. This approach is broadly applicable to gain a systems-level understanding of multifactorial 3D cell-matrix interactions.

  6. Crystal growth and electronic properties of a 3D Rashba material, BiTeI, with adjusted carrier concentrations.

    PubMed

    Kanou, Manabu; Sasagawa, Takao

    2013-04-03

    3D Rashba materials can be a leading player in spin-related novel phenomena, ranging from the metallic extreme (unconventional superconductivity) to the transport intermediate (spin Hall effects) to the novel insulating variant (3D topological insulating states). As the essential backbone for both fundamental and applied research of such a 3D Rashba material, this study established the growth of sizeable single crystals of a candidate compound BiTeI with adjusted carrier concentrations. Three techniques (standard vertical Bridgman, modified horizontal Bridgman, and vapour transport) were employed, and BiTeI crystals (>1 × 1 × 0.2 mm(3)) with fundamentally different electronic states from metallic to insulating were successfully grown by the chosen technique. The 3D Rashba electronic states, including the Fermi surface topology, for the corresponding carrier concentrations of the obtained BiTeI crystals were revealed by relativistic first-principles calculations.

  7. Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

    PubMed

    Escribano, J; Sánchez, M T; García-Aznar, J M

    2015-11-07

    Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding.

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

    PubMed

    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.

  9. 3D analysis of the performances degradation caused by series resistance in concentrator solar cells

    SciTech Connect

    Daliento, Santolo; Lancellotti, Laura

    2010-01-15

    This paper deals with the modeling of series resistance components in silicon concentrator solar cells. The main components of the macroscopic series resistance are analyzed by means of one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) numerical simulations. It is shown that the contribution of the lateral current flux, flowing along the emitter region, and of the transverse current flux, flowing along the metal grid, cannot be neglected and, hence, the operation of solar cells subjected to high current densities cannot be described by simple one-dimensional models. The percentage weight of 2D and 3D components on the total value of the series resistance is evaluated and rules for the proper design of the cell geometries are given. An analysis of the effectiveness of the most popular methods for the extraction of the series resistance from the I-V curves of solar cells is also proposed. (author)

  10. An approach to quantifying 3D responses of cells to extreme strain

    PubMed Central

    Li, Yuhui; Huang, Guoyou; Li, Moxiao; Wang, Lin; Elson, Elliot L.; Jian Lu, Tian; Genin, Guy M.; Xu, Feng

    2016-01-01

    The tissues of hollow organs can routinely stretch up to 2.5 times their length. Although significant pathology can arise if relatively large stretches are sustained, the responses of cells are not known at these levels of sustained strain. A key challenge is presenting cells with a realistic and well-defined three-dimensional (3D) culture environment that can sustain such strains. Here, we describe an in vitro system called microscale, magnetically-actuated synthetic tissues (micro-MASTs) to quantify these responses for cells within a 3D hydrogel matrix. Cellular strain-threshold and saturation behaviors were observed in hydrogel matrix, including strain-dependent proliferation, spreading, polarization, and differentiation, and matrix adhesion retained at strains sufficient for apoptosis. More broadly, the system shows promise for defining and controlling the effects of mechanical environment upon a broad range of cells. PMID:26887698

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-01

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

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

    PubMed

    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.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  16. Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake in 3D Cell Spheroids

    PubMed Central

    Rane, Tushar D.; Armani, Andrea M.

    2016-01-01

    Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy. PMID:27936027

  17. Using 3D Super-Resolution Microscopy to Probe Breast Cancer Stem Cells and Their Microenvironment

    DTIC Science & Technology

    2014-05-01

    microenvironments on breast cancer by creating arrays of polydimethlysiloxane (PDMS) microposts of different stiffness and sizes and seeded them with MCF-7 cells...of MCF-7s. Finally, with QPI, we investigated the real-time response of breast- cancer cells to different microenvironmental cues . We thus have...controls this cellular phenotype. To realize this goal, we had proposed to use 3D super-resolution microscopy to visualize how individual breast CaSCs

  18. A Cost-Effective Method to Assemble Biomimetic 3D Cell Culture Platforms

    PubMed Central

    Khalil, Sabreen; El-Badri, Nagwa; El-Mokhtaar, Mohamed; Al-Mofty, Saif; Farghaly, Mohamed; Ayman, Radwa; Habib, Dina; Mousa, Noha

    2016-01-01

    Developing effective stem cell based therapies requires the design of complex in vitro culture systems for more accurate representation of the stem cell niche. Attempts to improve conventional cell culture platforms include the use of biomaterial coated culture plates, sphere culture, microfluidic systems and bioreactors. Most of these platforms are not cost-effective, require industrial technical expertise to fabricate, and remain too simplistic compared to the physiological cell niche. The human amniotic membrane (hAM) has been used successfully in clinical grafting applications due to its unique biological composition and regenerative properties. In this study, we present a combinatorial platform that integrates the hAM with biomolecular, topographic and mechanical cues in one versatile model. Methods We utilized the hAM to provide the biological and the three dimensional (3D) topographic components of the prototype. The 3D nano-roughness of the hAM was characterized using surface electron microscopy and surface image analysis (ImageJ and SurfaceJ). We developed additional macro-scale and micro-scale versions of the platform which provided additional shear stress factors to simulate the fluid dynamics of the in vivo extracellular fluids. Results Three models of varying complexities of the prototype were assembled. A well-defined 3D surface modulation of the hAM in comparable to commercial 3D biomaterial culture substrates was achieved without complex fabrication and with significantly lower cost. Performance of the prototype was demonstrated through culture of primary human umbilical cord mononuclear blood cells (MNCs), human bone marrow mesenchymal stem cell line (hBMSC), and human breast cancer tissue. Conclusion This study presents methods of assembling an integrated, flexible and low cost biomimetic cell culture platform for diverse cell culture applications. PMID:27935982

  19. 3D Image-Guided Automatic Pipette Positioning for Single Cell Experiments in vivo

    PubMed Central

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

    2015-01-01

    We report a method to facilitate single cell, image-guided experiments including in vivo electrophysiology and electroporation. Our method combines 3D image data acquisition, visualization and on-line image analysis with precise control of physical probes such as electrophysiology microelectrodes in brain tissue in vivo. Adaptive pipette positioning provides a platform for future advances in automated, single cell in vivo experiments. PMID:26689553

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

    PubMed

    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.

  1. Cell Counting in Human Endobronchial Biopsies - Disagreement of 2D versus 3D Morphometry

    PubMed Central

    Bratu, Vlad A.; Erpenbeck, Veit J.; Fehrenbach, Antonia; Rausch, Tanja; Rittinghausen, Susanne; Krug, Norbert; Hohlfeld, Jens M.; Fehrenbach, Heinz

    2014-01-01

    Question Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D) counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D) methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel. Materials and Methods Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7) and smokers (n = 7), embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68) and T-lymphocytes (CD3), respectively. In identical fields of view, cell numbers per volume unit (NV) were assessed using the physical disector (3D), and profiles per area unit (NA) were counted (2D). For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis. Results In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05). When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups. Conclusions 2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a ‘universal conversion formula’. PMID:24663339

  2. A 3D engineered tumour for spatial snap-shot analysis of cell metabolism and phenotype in hypoxic gradients

    PubMed Central

    Rodenhizer, Darren; Gaude, Edoardo; Cojocari, Dan; Mahadevan, Radhakrishnan; Frezza, Christian; Wouters, Bradly G.; McGuigan, Alison P.

    2016-01-01

    The profound metabolic reprogramming that occurs in cancer cells has been investigated primarily in two-dimensional cell cultures, which fail to recapitulate spatial aspects of cell-to-cell interactions as well as tissue gradients present in three-dimensional (3D) tumours. Here, we describe an engineered model to assemble 3D tumours by rolling a scaffold-tumour composite strip. By unrolling the strip, the model can be rapidly disassembled for snap-shot analysis, allowing spatial mapping of cell metabolism in concert with cell phenotype. We also show that the establishment of oxygen gradients within samples are shaped by oxygen-dependent signalling pathways, as well as the consequential variations in cell growth, response to hypoxic gradients extending from normoxia to severe hypoxia, and therapy responsiveness, are consistent with tumours in vivo. Moreover, by using liquid chromatography tandem mass spectrometry, we mapped cellular metabolism and identified spatially defined metabolic signatures of cancer cells to reveal both known and novel metabolic responses to hypoxia. PMID:26595121

  3. Anticancer Drug Camptothecin Test in 3D Hydrogel Networks with HeLa cells

    PubMed Central

    Liang, Jun; Susan Sun, Xiuzhi; Yang, Zhilong; Cao, Shuai

    2017-01-01

    Development of a biomimetic 3D culture system for drug screening is necessary to fully understand the in vivo environment. Previously, a self-assembling peptide hydrogel has been reported; the hydrogel exhibited physiological properties superior to a 3D cell culture matrix. In this work, further research using H9e hydrogel with HeLa cells was carried out considering H9e hydrogel’s interaction with camptothecin, a hydrophobic drug. According to AFM images, a PGworks solution triggered H9e hydrogel fiber aggregation and forms a 3D matrix suitable for cell culture. Dynamic rheological studies showed that camptothecin was encapsulated within the hydrogel network concurrently with peptide self-assembly without permanently destroying the hydrogel’s architecture and remodeling ability. Fluorescence measurement indicated negligible interaction between the fluorophore part of camptothecin and the hydrogel, especially at concentration 0.25 and 0.5 wt%. Using a dialysis method, we found that H9e hydrogel could not significantly inhibit the diffusion of camptothecin encapsulated inside the hydrogel matrix. In the cell culture experiment, HeLa cells were simultaneously embedded in the H9e hydrogel with the initialization of hydrogelation. Most importantly, cell viability data after camptothecin treatment showed responses that were drug-dose dependent but unaffected by the H9e hydrogel concentration, indicating that the hydrogel did not inhibit the drug. PMID:28145436

  4. 3D cell culture: a review of current approaches and techniques.

    PubMed

    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.

  5. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed Central

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

    2013-01-01

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

  7. Behaviour of gravisensitive cells on 2D and 3D clinostats

    NASA Astrophysics Data System (ADS)

    Strauch, Sebastian M.; Hemmersbach, Ruth; Seibt, Dieter; Schuber, Marianne; Hader, Donat-P.

    2005-08-01

    2D and 3D clinostats are widely applied to study the influence of simulated microgravity on different kinds of organisms and cell cultures [1]. To critically evaluate the results achieved (functional weightlessness, omnilateral gravistimulation or other side effects such as strong mechanical disturbances) a comparison between the applied simulation methods and real microgravity is necessary. In a first approach, the swimming behavior of Euglena gracilis, a "professional gravi-sensing" unicellular freshwater flagellate, was observed under 2D and 3D clinostat conditions as well as under real microgravity during a TEXUS sounding rocket flight.According to current theory Euglena perceives the gravity vector by stimulation of mechanosensitive channels: the cell mass, which is denser than the surrounding medium, exerts pressure onto the lower membrane and the resulting gated calcium influx modulates the beating pattern of the flagella [4].A changed influence of gravity of the cells can be directly visualized by changes in their orientation with respect to gravity (gravitaxis).

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

    PubMed

    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.

  9. Thermo-responsive non-woven scaffolds for "smart" 3D cell culture.

    PubMed

    Rossouw, Claire L; Chetty, Avashnee; Moolman, Francis Sean; Birkholtz, Lyn-Marie; Hoppe, Heinrich; Mancama, Dalu T

    2012-08-01

    The thermo-responsive polymer poly(N-isopropylacrylamide) has received widespread attention for its in vitro application in the non-invasive, non-destructive release of adherent cells on two dimensional surfaces. In this study, 3D non-woven scaffolds fabricated from poly(propylene) (PP), poly(ethylene terephthalate) (PET), and nylon that had been grafted with PNIPAAm were tested for their ability to support the proliferation and subsequent thermal release of HC04 and HepG2 hepatocytes. Hepatocyte viability and proliferation were estimated using the Alamar Blue assay and Hoechst 33258 total DNA quantification. The assays revealed that the pure and grafted non-woven scaffolds maintained the hepatocytes within the matrix and promoted 3D proliferation comparable to that of the commercially available Algimatrix™ alginate scaffold. Albumin production and selected cytochrome P450 genes expression was found to be superior in cells growing on pure and grafted non-woven PP scaffolds as compared to cells grown as a 2D monolayer. Two scaffolds, namely, PP-g-PNIPAAm-A and PP-g-PNIPAAm-B were identified as having far superior thermal release capabilities; releasing the majority of the cells from the matrices within 2 h. This is the first report for the development of 3D non-woven, thermo-responsive scaffolds able to release cells from the matrix without the use of any enzymatic assistance or scaffold degradation.

  10. Establishment and Analysis of the 3-dimensional (3D) Spheroids Generated from the Nasopharyngeal Carcinoma Cell Line HK1

    PubMed Central

    Muniandy, Kalaivani; Sankar, Prabu Siva; Xiang, Benedict Lian Shi; Soo-Beng, Alan Khoo; Balakrishnan, Venugopal; Mohana-Kumaran, Nethia

    2016-01-01

    Spheroids have been shown to recapitulate the tumour in vivo with properties such as the tumour microenvironment, concentration gradients, and tumour phenotype. As such, it can serve as a platform for determining the growth and invasion behaviour pattern of the cancer cells as well as be utilised for drug sensitivity assays; capable of exhibiting results that are closer to what is observed in vivo compared to two-dimensional (2D) cell culture assays. This study focused on establishing a three-dimensional (3D) cell culture model using the Nasopharyngeal Carcinoma (NPC) cell line, HK1 and analysing its growth and invasion phenotypes. The spheroids will also serve as a model to elucidate their sensitivity to the chemotherapeutic drug, Flavopiridol. The liquid overlay method was employed to generate the spheroids which was embedded in bovine collagen I matrix for growth and invasion phenotypes observation. The HK1 cells formed compact spheroids within 72 hours. Our observation from the 3 days experiments revealed that the spheroids gradually grew and invaded into the collagen matrix, showing that the HK1 spheroids are capable of growth and invasion. Progressing from these experiments, the HK1 spheroids were employed to perform a drug sensitivity assay using the chemotherapeutic drug, Flavopiridol. The drug had a dose-dependent inhibition on spheroid growth and invasion. PMID:27965750

  11. 3D microfilter device for viable circulating tumor cell (CTC) enrichment from blood.

    PubMed

    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.

  12. Concentric gel system to study the biophysical role of matrix microenvironment on 3D cell migration.

    PubMed

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

    2015-04-03

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  15. A 3-D cardiac muscle construct for exploring adult marrow stem cell based myocardial regeneration.

    PubMed

    Valarmathi, Mani T; Goodwin, Richard L; Fuseler, John W; Davis, Jeffrey M; Yost, Michael J; Potts, Jay D

    2010-04-01

    Adult bone marrow stromal cells (BMSCs) are capable of differentiating into cardiomyocyte-like cells in vitro and contribute to myocardial regeneration in vivo. Consequently, BMSCs may potentially play a vital role in cardiac repair and regeneration. However, this concept has been limited by inadequate and inconsistent differentiation of BMSCs into cardiomyocytes along with poor survival and integration of neo-cardiomyocytes after implantation into ischemic myocardium. In order to overcome these barriers and to explore adult stem cell based myocardial regeneration, we have developed an in vitro model of three-dimensional (3-D) cardiac muscle using rat ventricular embryonic cardiomyocytes (ECMs) and BMSCs. When ECMs and BMSCs were seeded sequentially onto a 3-D tubular scaffold engineered from topographically aligned type I collagen-fibers and cultured in basal medium for 7, 14, 21, or 28 days, the maturation and co-differentiation into a cardiomyocyte lineage was observed. Phenotypic induction was characterized at morphological, immunological, biochemical and molecular levels. The observed expression of transcripts coding for cardiomyocyte phenotypic markers and the immunolocalization of cardiomyogenic lineage-associated proteins revealed typical expression patterns of neo-cardiomyogenesis. At the biochemical level differentiating cells exhibited appropriate metabolic activity and at the ultrastructural level myofibrillar and sarcomeric organization were indicative of an immature phenotype. Our 3-D co-culture system sustains the ECMs in vitro continuum of differentiation process and simultaneously induces the maturation and differentiation of BMSCs into cardiomyocyte-like cells. Thus, this novel 3-D co-culture system provides a useful in vitro model to investigate the functional role and interplay of developing ECMs and BMSCs during cardiomyogenic differentiation.

  16. Kinematics of a growth fault/raft system on the West African margin using 3-D restoration

    NASA Astrophysics Data System (ADS)

    Rouby, Delphine; Raillard, Stéphane; Guillocheau, François; Bouroullec, Renaud; Nalpas, Thierry

    2002-04-01

    The ability to quantify the movement history associated with growth structures is crucial in the understanding of fundamental processes such as the growth of folds or faults in 3-D. In this paper, we present an application of an original approach to restore in 3-D a listric growth fault system resulting from gravity-induced extension located on the West African margin. Our goal is to establish the 3-D structural framework and kinematics of the study area. We construct a 3-D geometrical model of the fault system (from 3-D seismic data), then restore six stratigraphic surfaces and reconstruct the 3-D geometry of the system at six incremental steps of its history. The evolution of the growth fault/raft system corresponds to the progressive separation of two rafts by regional extension, resulting in the development of an intervening basin located between them that evolved in three main stages: (1) the rise of an evaporite wall, (2) the development of a symmetric basin as the elevation of the diapir is reduced and buried, and (3) the development of asymmetric basins related to two systems of listric faults (the main fault F1 and the graben located between the rollovers and the lower raft). Important features of the growth fault/raft system could only be observed in 3-D and with increments of deformation restored. The rollover anticline (associated with the listric fault F1) is composed of two sub-units separated by an E-W oriented transverse graben indicating that the displacement field was divergent in map view. The rollover units are located within the overlap area of two fault systems and displays a 'mock-turtle' anticline structure. The seaward translation of the lower raft is associated with two successive vertical axis rotations in the opposite sense (clockwise then counter-clockwise by about 10°). This results from the fact that the two main fault systems developed successively. Fault system F1 formed during the Upper Albian, and the graben during the Cenomanian

  17. Quantitative 3D Tracing of Gene-delivery Viral Vectors in Human Cells and Animal Tissues

    PubMed Central

    Xiao, Ping-Jie; Li, Chengwen; Neumann, Aaron; Samulski, R Jude

    2012-01-01

    Trafficking through a variety of cellular structures and organelles is essential for the interaction between gene-delivery vectors (i.e., adeno-associated virus (AAV) and liposomes) and host cells/tissues. Here, we present a method of computer-assisted quantitative 3D biodistribution microscopy that samples the whole population of fluorescently-labeled vectors and document their trafficking routes. Using AAV as a working model, we first experimentally defined numerical parameters for the singularity of Cy5-labeled particles by combining confocal microscopy and atomic force microscopy (AFM). We then developed a robust approach that integrates single-particle fluorescence imaging with 3D deconvolution and isosurface rendering to quantitate viral distribution and trafficking in human cells as well as animal tissues at the single-particle level. Using this quantitative method, we uncovered an as yet uncharacterized rate-limiting step during viral cell entry, while delineating nuclear accumulation of virions during the first 8 hours postinfection. Further, our studies revealed for the first time that following intramuscular injection, AAV spread progressively across muscle tissues through endomysium between myofibers instead of traversing through target cells. Such 3D resolution and quantitative dissection of vector–host interactions at the subcellular level should significantly improve our ability to resolve trafficking mechanisms of gene-delivery particles and facilitate the development of enhanced viral vectors. PMID:22108857

  18. Comparative mechanisms of cancer cell migration through 3D matrix and physiological microtracks.

    PubMed

    Carey, Shawn P; Rahman, Aniqua; Kraning-Rush, Casey M; Romero, Bethsabe; Somasegar, Sahana; Torre, Olivia M; Williams, Rebecca M; Reinhart-King, Cynthia A

    2015-03-15

    Tumor cell invasion through the stromal extracellular matrix (ECM) is a key feature of cancer metastasis, and understanding the cellular mechanisms of invasive migration is critical to the development of effective diagnostic and therapeutic strategies. Since cancer cell migration is highly adaptable to physiochemical properties of the ECM, it is critical to define these migration mechanisms in a context-specific manner. Although extensive work has characterized cancer cell migration in two- and three-dimensional (3D) matrix environments, the migration program employed by cells to move through native and cell-derived microtracks within the stromal ECM remains unclear. We previously reported the development of an in vitro model of patterned type I collagen microtracks that enable matrix metalloproteinase-independent microtrack migration. Here we show that collagen microtracks closely resemble channel-like gaps in native mammary stroma ECM and examine the extracellular and intracellular mechanisms underlying microtrack migration. Cell-matrix mechanocoupling, while critical for migration through 3D matrix, is not necessary for microtrack migration. Instead, cytoskeletal dynamics, including actin polymerization, cortical tension, and microtubule turnover, enable persistent, polarized migration through physiological microtracks. These results indicate that tumor cells employ context-specific mechanisms to migrate and suggest that selective targeting of cytoskeletal dynamics, but not adhesion, proteolysis, or cell traction forces, may effectively inhibit cancer cell migration through preformed matrix microtracks within the tumor stroma.

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

    PubMed Central

    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

  20. Tuning cell adhesion by controlling the roughness and wettability of 3D micro/nano silicon structures.

    PubMed

    Ranella, A; Barberoglou, M; Bakogianni, S; Fotakis, C; Stratakis, E

    2010-07-01

    The aim of this study is to investigate fibroblast cell adhesion and viability on highly rough three-dimensional (3D) silicon (Si) surfaces with gradient roughness ratios and wettabilities. Culture surfaces were produced by femtosecond (fs) laser structuring of Si wafers and comprised forests of conical spikes exhibiting controlled dual-scale roughness at both the micro- and the nano-scale. Variable roughness could be achieved by changing the laser pulse fluence and control over wettability and therefore surface energy could be obtained by covering the structures with various conformal coatings, which altered the surface chemistry without, however, affecting morphology. The results showed that optimal cell adhesion was obtained for small roughness ratios, independently of the surface wettability and chemistry, indicating a non-monotonic dependence of fibroblast adhesion on surface energy. Additionally, it was shown that, for the same degree of roughness, a proper change in surface energy could switch the behaviour from cell-phobic to cell-philic and vice versa, transition that was always correlated to surface wettability. These experimental findings are discussed on the basis of previous theoretical models describing the relation of cell response to surface energy. The potential use of the patterned Si substrates as model scaffolds for the systematic exploration of the role of 3D micro/nano morphology and/or surface energy on cell adhesion and growth is envisaged.

  1. n-ZnO/p-Si 3D heterojunction solar cells in Si holey arrays

    NASA Astrophysics Data System (ADS)

    Zhang, Xiao-Mei; Golberg, Dmitri; Bando, Yoshio; Fukata, Naoki

    2012-01-01

    A wafer-scale, low-cost solar cell based on n-ZnO/p-Si 3D heterojunction arrays on holey Si substrates has been fabricated. This device shows a power-conversion efficiency of 1.2% and high photosensitivity. The present n-ZnO/p-Si heterojunction architectures are envisaged as potentially valuable candidates for next-generation photovoltaics.A wafer-scale, low-cost solar cell based on n-ZnO/p-Si 3D heterojunction arrays on holey Si substrates has been fabricated. This device shows a power-conversion efficiency of 1.2% and high photosensitivity. The present n-ZnO/p-Si heterojunction architectures are envisaged as potentially valuable candidates for next-generation photovoltaics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11752e

  2. Architectural proteins: Regulators of 3D genome organization in cell fate

    PubMed Central

    Gómez-Díaz, Elena; Corces, Victor G.

    2014-01-01

    The relationship between alterations in chromatin structure and changes in gene expression during cell differentiation has served as a paradigm to understand the link between genome organization and function. Yet the factors involved and the mechanisms by which the three-dimensional organization of the nucleus is established remain poorly understood. The use of Chromosome Conformation-Capture (3C) based approaches has resulted in a new appreciation of the role of architectural proteins in the establishment of 3D genome organization. Architectural proteins orchestrate higher-order chromatin organization through the establishment of interactions between regulatory elements across multiple spatial scales. The regulation of these proteins, their interaction with DNA, and their co occurrence in the genome, may be responsible for the plasticity of 3D-chromatin architecture that dictates cell and time-specific blueprints of gene expression. PMID:25218583

  3. Next-generation regenerative medicine: organogenesis from stem cells in 3D culture.

    PubMed

    Sasai, Yoshiki

    2013-05-02

    The behavior of stem cells, when they work collectively, can be much more sophisticated than one might expect from their individual programming. This Perspective covers recent discoveries about the dynamic patterning and structural self-formation of complex organ buds in 3D stem cell culture, including the generation of various neuroectodermal and endodermal tissues. For some tissues, epithelial-mesenchymal interactions can also be manipulated in coculture to guide organogenesis. This new area of stem cell research-the spatiotemporal control of dynamic cellular interactions-will open a new avenue for next-generation regenerative medicine.

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

    SciTech Connect

    Roche, P.; Palau, J.M.; Bruguier, G.; Tavernier, C.; Ecoffet, R.; Gasiot, J.

    1999-12-01

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

  5. Coculture system with an organotypic brain slice and 3D spheroid of carcinoma cells.

    PubMed

    Chuang, Han-Ning; Lohaus, Raphaela; Hanisch, Uwe-Karsten; Binder, Claudia; Dehghani, Faramarz; Pukrop, Tobias

    2013-10-09

    Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis(1,2). Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation(3). Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells(4,5). Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis(6,7). Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior(8). However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible(8). For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to

  6. Monolithic cell counter based on 3D hydrodynamic focusing in microfluidic channels

    NASA Astrophysics Data System (ADS)

    Paiè, Petra; Bragheri, Francesca; Osellame, Roberto

    2014-03-01

    Hydrodynamic focusing is a powerful technique frequently used in microfluidics that presents a wide range of applications since it allows focusing the sample flowing in the device to a narrow region in the center of the microchannel. In fact thanks to the laminarity of the fluxes in microchannels it is possible to confine the sample solution with a low flow rate by using a sheath flow with a higher flow rate. This in turn allows the flowing of one sample element at a time in the detection region, thus enabling analysis on single particles. Femtosecond laser micromachining is ideally suited to fabricate device integrating full hydrodynamic focusing functionalities thanks to the intrinsic 3D nature of this technique, especially if compared to expensive and complicated lithographic multi-step fabrication processes. Furthermore, because of the possibility to fabricate optical waveguides with the same technology, it is possible to obtain compact optofluidic devices to perform optical analysis of the sample even at the single cell level, as is the case for optical cell stretchers and sorters. In this work we show the fabrication and the fluidic characterization of extremely compact devices having only two inlets for 2D (both in vertical and horizontal planes) as well as full 3D symmetric hydrodynamic focusing. In addition we prove one of the possible application of the hydrodynamic focusing module, by fabricating and validating (both with polystyrene beads and erythrocytes) a monolithic cell counter obtained by integrating optical waveguides in the 3D hydrodynamic focusing device.

  7. Nucleus and nucleus-cytoskeleton connections in 3D cell migration.

    PubMed

    Liu, Lingling; Luo, Qing; Sun, Jinghui; Song, Guanbin

    2016-10-15

    Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and review how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration.

  8. Space-time evolution of a growth fold (Betic Cordillera, Spain). Evidences from 3D geometrical modelling

    NASA Astrophysics Data System (ADS)

    Martin-Rojas, Ivan; Alfaro, Pedro; Estévez, Antonio

    2014-05-01

    We present a study that encompasses several software tools (iGIS©, ArcGIS©, Autocad©, etc.) and data (geological mapping, high resolution digital topographic data, high resolution aerial photographs, etc.) to create a detailed 3D geometric model of an active fault propagation growth fold. This 3D model clearly shows structural features of the analysed fold, as well as growth relationships and sedimentary patterns. The results obtained permit us to discuss the kinematics and structural evolution of the fold and the fault in time and space. The study fault propagation fold is the Crevillente syncline. This fold represents the northern limit of the Bajo Segura Basin, an intermontane basin in the Eastern Betic Cordillera (SE Spain) developed from upper Miocene on. 3D features of the Crevillente syncline, including growth pattern, indicate that limb rotation and, consequently, fault activity was higher during Messinian than during Tortonian; consequently, fault activity was also higher. From Pliocene on our data point that limb rotation and fault activity steadies or probably decreases. This in time evolution of the Crevillente syncline is not the same all along the structure; actually the 3D geometric model indicates that observed lateral heterogeneity is related to along strike variation of fault displacement.

  9. Economic 3D-printing approach for transplantation of human stem cell-derived β-like cells.

    PubMed

    Song, Jiwon; Millman, Jeffrey R

    2016-12-01

    Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing β cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived β (SC-β) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-β cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-β cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.

  10. 3D Cell Entrapment as a Function of the Weight Percent of Peptide-Amphiphile Hydrogels

    PubMed Central

    Scott, Carolyn M.; Forster, Colleen L.; Kokkoli, Efrosini

    2015-01-01

    The design of scaffolds which mimic the stiffness, nanofiber structure, and biochemistry of the native extra-cellular matrix (ECM) has been a major objective for the tissue engineering field. Furthermore, mimicking the innate three dimensional (3D) environment of the ECM has been shown to significantly alter cellular response compared to traditional two dimensional (2D) culture. We report the development of a self-assembling, fibronectin-mimetic, peptide-amphiphile nanofiber scaffold for 3D cell culture. To form such a scaffold, 5 mol% of a bioactive PR_g fibronectin-mimetic peptide-amphiphile was mixed with 95 mol% of a diluent peptide-amphiphile (E2) whose purpose was to neutralize electrostatic interactions, increase the gelation kinetics and promote cell survival. Atomic force microscopy verified the fibrilar structure of the gels and the mechanical properties were characterized for various weight percent (wt%) formulations of the 5 mol% PR_g - 95 mol% E2 peptide-amphiphile mixture. The 0.5 wt% formulations had an elastic modulus of 429.0 ± 21.3 Pa while the 1.0 wt% peptide-amphiphile hydrogels had an elastic modulus of 808.6 ± 38.1 Pa. The presence of entrapped cells in the gels decreased the elastic modulus and the decrease was a function of the cell loading. While both formulations supported cell proliferation, the 0.5 wt% gels supported significantly greater NIH3T3/GFP fibroblast cell proliferation throughout the gels than the 1.0 wt% gels. However, compared to the 0.5 wt% formulations, the 1.0 wt% hydrogels promoted greater increase in mRNA expression and production of fibronectin and type IV collagen ECM proteins. This study suggests that this fibronectin-mimetic scaffold holds great promise in the advance of 3D culture applications and cell therapies. PMID:25970351

  11. VA-086 methacrylate gelatine photopolymerizable hydrogels: A parametric study for highly biocompatible 3D cell embedding.

    PubMed

    Occhetta, Paola; Visone, Roberta; Russo, Laura; Cipolla, Laura; Moretti, Matteo; Rasponi, Marco

    2015-06-01

    The ability to replicate in vitro the native extracellular matrix (ECM) features and to control the three-dimensional (3D) cell organization plays a fundamental role in obtaining functional engineered bioconstructs. In tissue engineering (TE) applications, hydrogels have been successfully implied as biomatrices for 3D cell embedding, exhibiting high similarities to the natural ECM and holding easily tunable mechanical properties. In the present study, we characterized a promising photocrosslinking process to generate cell-laden methacrylate gelatin (GelMA) hydrogels in the presence of VA-086 photoinitiator using a ultraviolet LED source. We investigated the influence of prepolymer concentration and light irradiance on mechanical and biomimetic properties of resulting hydrogels. In details, the increasing of gelatin concentration resulted in enhanced rheological properties and shorter polymerization time. We then defined and validated a reliable photopolymerization protocol for cell embedding (1.5% VA-086, LED 2 mW/cm2) within GelMA hydrogels, which demonstrated to support bone marrow stromal cells viability when cultured up to 7 days. Moreover, we showed how different mechanical properties, derived from different crosslinking parameters, strongly influence cell behavior. In conclusion, this protocol can be considered a versatile tool to obtain biocompatible cell-laden hydrogels with properties easily adaptable for different TE applications.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    PubMed

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

    2015-07-10

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

  16. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

    PubMed Central

    Jeon, Jessie S.; Bersini, Simone; Gilardi, Mara; Dubini, Gabriele; Charest, Joseph L.; Moretti, Matteo; Kamm, Roger D.

    2015-01-01

    A key aspect of cancer metastases is the tendency for specific cancer cells to home to defined subsets of secondary organs. Despite these known tendencies, the underlying mechanisms remain poorly understood. Here we develop a microfluidic 3D in vitro model to analyze organ-specific human breast cancer cell extravasation into bone- and muscle-mimicking microenvironments through a microvascular network concentrically wrapped with mural cells. Extravasation rates and microvasculature permeabilities were significantly different in the bone-mimicking microenvironment compared with unconditioned or myoblast containing matrices. Blocking breast cancer cell A3 adenosine receptors resulted in higher extravasation rates of cancer cells into the myoblast-containing matrices compared with untreated cells, suggesting a role for adenosine in reducing extravasation. These results demonstrate the efficacy of our model as a drug screening platform and a promising tool to investigate specific molecular pathways involved in cancer biology, with potential applications to personalized medicine. PMID:25524628

  17. 3D Bioprinting of complex channels-Effects of material, orientation, geometry, and cell embedding.

    PubMed

    Wüst, Silke; Müller, Ralph; Hofmann, Sandra

    2015-08-01

    Creating filled or hollow channels within 3D tissues has become increasingly important in tissue engineering. Channels can serve as vasculature enhancing medium perfusion or as conduits for nerve regeneration. The 3D biofabrication seems to be a promising method to generate these structures within 3D constructs layer-by-layer. In this study, geometry and interface of bioprinted channels were investigated with micro-computed tomography and fluorescent imaging. In filament printing, size and shape of printed channels are influenced by their orientation, which was analyzed by printing horizontally and vertically aligned channels, and by the ink, which was evaluated by comparing channels printed with an alginate-gelatin hydrogel or with an emulsion. The influence of geometry and cell-embedding in the hydrogel on feature size and shape was investigated by printing more complex channels. The generation of hollow channels, induced through leaching of a support phase, was monitored over time. Horizontally aligned channels provided 16× smaller cross-sectional areas than channels in vertical orientation. The smallest feature size of hydrogel filaments was twice as large compared to emulsion filaments. Feature size and shape depended on the geometry but did not alter when living cells were embedded. With that knowledge, channels can be consciously tailored to the particular needs.

  18. Boosting Power Density of Microbial Fuel Cells with 3D Nitrogen-Doped Graphene Aerogel Electrode.

    PubMed

    Yang, Yang; Liu, Tianyu; Zhu, Xun; Zhang, Feng; Ye, Dingding; Liao, Qiang; Li, Yat

    2016-08-01

    A 3D nitrogen-doped graphene aerogel (N-GA) as an anode material for microbial fuel cells (MFCs) is reported. Electron microscopy images reveal that the N-GA possesses hierarchical porous structure that allows efficient diffusion of both bacterial cells and electron mediators in the interior space of 3D electrode, and thus, the colonization of bacterial communities. Electrochemical impedance spectroscopic measurements further show that nitrogen doping considerably reduces the charge transfer resistance and internal resistance of GA, which helps to enhance the MFC power density. Importantly, the dual-chamber milliliter-scale MFC with N-GA anode yields an outstanding volumetric power density of 225 ± 12 W m(-3) normalized to the total volume of the anodic chamber (750 ± 40 W m(-3) normalized to the volume of the anode). These power densities are the highest values report for milliliter-scale MFCs with similar chamber size (25 mL) under the similar measurement conditions. The 3D N-GA electrode shows great promise for improving the power generation of MFC devices.

  19. Boosting Power Density of Microbial Fuel Cells with 3D Nitrogen‐Doped Graphene Aerogel Electrode

    PubMed Central

    Yang, Yang; Liu, Tianyu; Zhang, Feng; Ye, Dingding; Liao, Qiang

    2016-01-01

    A 3D nitrogen‐doped graphene aerogel (N‐GA) as an anode material for microbial fuel cells (MFCs) is reported. Electron microscopy images reveal that the N‐GA possesses hierarchical porous structure that allows efficient diffusion of both bacterial cells and electron mediators in the interior space of 3D electrode, and thus, the colonization of bacterial communities. Electrochemical impedance spectroscopic measurements further show that nitrogen doping considerably reduces the charge transfer resistance and internal resistance of GA, which helps to enhance the MFC power density. Importantly, the dual‐chamber milliliter‐scale MFC with N‐GA anode yields an outstanding volumetric power density of 225 ± 12 W m−3 normalized to the total volume of the anodic chamber (750 ± 40 W m−3 normalized to the volume of the anode). These power densities are the highest values report for milliliter‐scale MFCs with similar chamber size (25 mL) under the similar measurement conditions. The 3D N‐GA electrode shows great promise for improving the power generation of MFC devices. PMID:27818911

  20. Accurate Automatic Detection of Densely Distributed Cell Nuclei in 3D Space

    PubMed Central

    Tokunaga, Terumasa; Kanamori, Manami; Teramoto, Takayuki; Jang, Moon Sun; Kuge, Sayuri; Ishihara, Takeshi; Yoshida, Ryo; Iino, Yuichi

    2016-01-01

    To measure the activity of neurons using whole-brain activity imaging, precise detection of each neuron or its nucleus is required. In the head region of the nematode C. elegans, the neuronal cell bodies are distributed densely in three-dimensional (3D) space. However, no existing computational methods of image analysis can separate them with sufficient accuracy. Here we propose a highly accurate segmentation method based on the curvatures of the iso-intensity surfaces. To obtain accurate positions of nuclei, we also developed a new procedure for least squares fitting with a Gaussian mixture model. Combining these methods enables accurate detection of densely distributed cell nuclei in a 3D space. The proposed method was implemented as a graphical user interface program that allows visualization and correction of the results of automatic detection. Additionally, the proposed method was applied to time-lapse 3D calcium imaging data, and most of the nuclei in the images were successfully tracked and measured. PMID:27271939

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  4. Rapid 3D Refractive‐Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation

    PubMed Central

    Habaza, Mor; Kirschbaum, Michael; Guernth‐Marschner, Christian; Dardikman, Gili; Barnea, Itay; Korenstein, Rafi; Duschl, Claus

    2016-01-01

    A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label‐free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive‐index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive‐index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label‐free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label‐free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids. PMID:28251046

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

    NASA Astrophysics Data System (ADS)

    Bunga, Pratap Kumar

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

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

    SciTech Connect

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    PubMed

    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.

  9. In the beginning there were soft collagen-cell gels: towards better 3D connective tissue models?

    PubMed

    Brown, Robert A

    2013-10-01

    In the 40 years since Elsdale and Bard's analysis of fibroblast culture in collagen gels we have moved far beyond the concept that such 3D fibril network systems are better models than monolayer cultures. This review analyses key aspects of that progression of models, against a background of what exactly each model system tries to mimic. This story tracks our increasing understanding of fibroblast responses to soft collagen gels, in particularly their cytoskeletal contraction, migration and integrin attachment. The focus on fibroblast mechano-function has generated models designed to directly measure the overall force generated by fibroblast populations, their reaction to external loads and the role of the matrix structure. Key steps along this evolution of 3D collagen models have been designed to mimic normal skin, wound repair, tissue morphogenesis and remodelling, growth and contracture during scarring/fibrosis. As new models are developed to understand cell-mechanical function in connective tissues the collagen material has become progressively more important, now being engineered to mimic more complex aspects of native extracellular matrix structure. These have included collagen fibril density, alignment and hierarchical structure, controlling material stiffness and anisotropy. But of these, tissue-like collagen density is key in that it contributes to control of the others. It is concluded that across this 40 year window major progress has been made towards establishing a family of 3D experimental collagen tissue-models, suitable to investigate normal and pathological fibroblast mechano-functions.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27248849

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

    PubMed Central

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

    2009-01-01

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

  13. Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture

    PubMed Central

    Gieseck III, Richard L.; Hannan, Nicholas R. F.; Bort, Roque; Hanley, Neil A.; Drake, Rosemary A. L.; Cameron, Grant W. W.; Wynn, Thomas A.; Vallier, Ludovic

    2014-01-01

    Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard. PMID:24466060

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

    NASA Astrophysics Data System (ADS)

    Danilevicius, Paulius; Georgiadi, Leoni; Pateman, Christopher J.; Claeyssens, Frederik; Chatzinikolaidou, Maria; Farsari, Maria

    2015-05-01

    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.

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

    PubMed

    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.

  16. In Situ "Clickable" Zwitterionic Starch-Based Hydrogel for 3D Cell Encapsulation.

    PubMed

    Dong, Dianyu; Li, Junjie; Cui, Man; Wang, Jinmei; Zhou, Yuhang; Luo, Liu; Wei, Yufei; Ye, Lei; Sun, Hong; Yao, Fanglian

    2016-02-01

    Three-dimensional (3D) cell encapsulation in hydrogel provides superb methods to investigate the biochemical cues in directing cellular fate and behaviors outside the organism, the primary step of which is to establish suitable "blank platform" to mimic and simplify native ECM microenvironment. In this study, zwitterionic starch-based "clickable" hydrogels were fabricated via a "copper- and light- free" Michael-type "thiol-ene" addition reaction between acylated-modified sulfobetaine-derived starch (SB-ST-A) and dithiol-functionalized poly(ethylene glycol) (PEG-SH). By incorporating antifouling SB-ST and PEG, the hydrogel system would be excellently protected from nontarget protein adsorption to act as a "blank platform". The hydrogels could rapidly gel under physiological conditions in less than 7 min. Dynamic rheology experiments suggested the stiffness of the hydrogel was close to the native tissues, and the mechanical properties as well as the gelation times and swelling behaviors could be easily tuned by varying the precursor proportions. The protein and cell adhesion assays demonstrated that the hydrogel surface could effectively resist nonspecific protein and cell adhesion. The degradation study in vitro confirmed that the hydrogel was biodegradable. A549 cells encapsulated in the hydrogel maintained high viability (up to 93%) and started to proliferate in number and extend in morphology after 2 days' culture. These results indicated the hydrogel presented here could be a potential candidate as "blank platform" for 3D cell encapsulation and biochemical cues induced cellular behavior investigation in vitro.

  17. Pluripotent stem cell expansion and neural differentiation in 3-D scaffolds of tunable Poisson's ratio.

    PubMed

    Yan, Yuanwei; Li, Yan; Song, Liqing; Zeng, Changchun; Li, Yan

    2017-02-01

    Biophysical properties of the scaffolds such as the elastic modulus, have been recently shown to impact stem cell lineage commitment. On the other hand, the contribution of the Poisson's ratio, another important biophysical property, to the stem cell fate decision, has not been studied. Scaffolds with tunable Poisson's ratio (ν) (termed as auxetic scaffolds when Poisson's ratio is zero or negative) are anticipated to provide a spectrum of unique biophysical 3-D microenvironments to influence stem cell fate. To test this hypothesis, in the present work we fabricated auxetic polyurethane scaffolds (ν=0 to -0.45) and evaluated their effects on neural differentiation of mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (hiPSCs). Compared to the regular scaffolds (ν=+0.30) before auxetic conversion, the auxetic scaffolds supported smaller aggregate formation and higher expression of β-tubulin III upon neural differentiation. The influences of pore structure, Poisson's ratio, and elastic modulus on neural lineage commitment were further evaluated using a series of auxetic scaffolds. The results indicate that Poisson's ratio may confound the effects of elastic modulus, and auxetic scaffolds with proper pore structure and Poisson's ratio enhance neural differentiation. This study demonstrates that tuning the Poisson's ratio of the scaffolds together with elastic modulus and microstructure would enhance the capability to generate broader, more diversified ranges of biophysical 3-D microenvironments for the modulation of cellular differentiation.

  18. The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II.

    PubMed

    Jean, Léolène; Yang, Lijie; Majumdar, Devi; Gao, Yandong; Shi, Mingjian; Brewer, Bryson M; Li, Deyu; Webb, Donna J

    2014-01-01

    Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.

  19. ECM Composition and Rheology Regulate Growth, Motility, and Response to Photodynamic Therapy in 3D Models of Pancreatic Ductal Adenocarcinoma.

    PubMed

    Cramer, Gwendolyn M; Jones, Dustin P; El-Hamidi, Hamid; Celli, Jonathan P

    2017-01-01

    Pancreatic ductal adenocarcinoma is characterized by prominent stromal involvement, which plays complex roles in regulating tumor growth and therapeutic response. The extracellular matrix (ECM)-rich stroma associated with this disease has been implicated as a barrier to drug penetration, although stromal depletion strategies have had mixed clinical success. It remains less clear how interactions with ECM, acting as a biophysical regulator of phenotype, not only a barrier to drug perfusion, regulate susceptibilities and resistance to specific therapies. In this context, an integrative approach is used to evaluate invasive behavior and motility in rheologically characterized ECM as determinants of chemotherapy and photodynamic therapy (PDT) responses. We show that in 3D cultures with ECM conditions that promote invasive progression, response to PDT is markedly enhanced in the most motile ECM-infiltrating populations, whereas the same cells exhibit chemoresistance. Conversely, drug-resistant sublines with enhanced invasive potential were generated to compare differential treatment response in identical ECM conditions, monitored by particle tracking microrheology measurements of matrix remodeling. In both scenarios, ECM-infiltrating cell populations exhibit increased sensitivity to PDT, whether invasion is consequent to selection of chemoresistance, or whether chemoresistance is correlated with acquisition of invasive behavior. However, while ECM-invading, chemoresistant cells exhibit mesenchymal phenotype, induction of EMT in monolayers without ECM was not sufficient to enhance PDT sensitivity, yet does impart chemoresistance as expected. In addition to containing platform development with broader applicability to inform microenvironment-dependent therapeutics, these results reveal the efficacy of PDT for targeting the most aggressive, chemoresistant, invasive pancreatic ductal adenocarcinoma associated with dismal outcomes for this disease.

  20. Human bronchial epithelial cells differentiate to 3D glandular acini on basement membrane matrix.

    PubMed

    Wu, Xiaofang; Peters-Hall, Jennifer R; Bose, Sumit; Peña, Maria T; Rose, Mary C

    2011-06-01

    To create a model system that investigates mechanisms resulting in hyperplasia and hypertrophy of respiratory tract submucosal glands, we developed an in vitro three-dimensional (3D) system wherein normal human bronchial epithelial (HBE) cells differentiated into glandular acini when grown on a basement membrane matrix. The differentiation of primary HBE cells into glandular acini was monitored temporally by light microscopy. Apoptosis-induced lumen formation was observed by immunofluorescence analysis. The acinar cells expressed and secreted MUC5B mucin (marker for glandular mucous cells) and lysozyme, lactoferrin, and zinc-α2-glycoprotein (markers for glandular serous cells) at Day 22. β-Tubulin IV, a marker for ciliated cells, was not detected. Expression of mucous and serous cell markers in HBE glandular acini demonstrated that HBE cells grown on a basement membrane matrix differentiated into acini that exhibit molecular characteristics of respiratory tract glandular acinar cells. Inhibition studies with neutralizing antibodies resulted in a marked decrease in size of the spheroids at Day 7, demonstrating that laminin (a major component of the basement membrane matrix), the cell surface receptor integrin α6, and the cell junction marker E-cadherin have functional roles in HBE acinar morphogenesis. No significant variability was detected in the average size of glandular acini formed by HBE cells from two normal individuals. These results demonstrated that this in vitro model system is reproducible, stable, and potentially useful for studies of glandular differentiation and hyperplasia.

  1. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    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.

  2. Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads.

    PubMed

    Wang, Lin; Acosta, Miguel A; Leach, Jennie B; Carrier, Rebecca L

    2013-04-21

    Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.

  3. Spacecraft charging analysis with the implicit particle-in-cell code iPic3D

    SciTech Connect

    Deca, J.; Lapenta, G.; Marchand, R.; Markidis, S.

    2013-10-15

    We present the first results on the analysis of spacecraft charging with the implicit particle-in-cell code iPic3D, designed for running on massively parallel supercomputers. The numerical algorithm is presented, highlighting the implementation of the electrostatic solver and the immersed boundary algorithm; the latter which creates the possibility to handle complex spacecraft geometries. As a first step in the verification process, a comparison is made between the floating potential obtained with iPic3D and with Orbital Motion Limited theory for a spherical particle in a uniform stationary plasma. Second, the numerical model is verified for a CubeSat benchmark by comparing simulation results with those of PTetra for space environment conditions with increasing levels of complexity. In particular, we consider spacecraft charging from plasma particle collection, photoelectron and secondary electron emission. The influence of a background magnetic field on the floating potential profile near the spacecraft is also considered. Although the numerical approaches in iPic3D and PTetra are rather different, good agreement is found between the two models, raising the level of confidence in both codes to predict and evaluate the complex plasma environment around spacecraft.

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

    PubMed

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

    2016-04-21

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed

    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.

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

    PubMed

    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.

  8. 3D bioprinting of biomimetic aortic vascular constructs with self-supporting cells.

    PubMed

    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.

  9. Advances in automated 3-D image analyses of cell populations imaged by confocal microscopy.

    PubMed

    Ancin, H; Roysam, B; Dufresne, T E; Chestnut, M M; Ridder, G M; Szarowski, D H; Turner, J N

    1996-11-01

    Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

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

    PubMed

    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.

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

    PubMed

    Olsen, Mark Holm; Hjortø, Gertrud Malene; Hansen, Morten; Met, Özcan; Svane, Inge Marie; Larsen, Niels B

    2013-12-21

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  14. A Novel Core-Shell Microcapsule for Encapsulation and 3D Culture of Embryonic Stem Cells

    PubMed Central

    Zhang, Wujie; Zhao, Shuting; Rao, Wei; Snyder, Jedidiah; Choi, Jung K.; Wang, Jifu; Khan, Iftheker A.; Saleh, Navid B.; Mohler, Peter J.; Yu, Jianhua; Hund, Thomas J.; Tang, Chuanbing; He, Xiaoming

    2013-01-01

    In this study, we report the preparation of a novel microcapsule of ~ 100 μm with a liquid (as compared to solid-like alginate hydrogel) core and an alginate-chitosan-alginate (ACA) shell for encapsulation and culture of embryonic stem (ES) cells in the miniaturized 3D space of the liquid core. Murine R1 ES cells cultured in the microcapsules were found to survive (> 90%) well and proliferate to form either a single aggregate of pluripotent cells or embryoid body (EB) of more differentiated cells in each microcapsule within 7 days, dependent on the culture medium used. This novel microcapsule technology allows massive production of the cell aggregates or EBs of uniform size and controllable pluripotency, which is important for the practical application of stem cell based therapy. Moreover, the semipermeable ACA shell was found to significantly reduce immunoglobulin G (IgG) binding to the encapsulated cells by up to 8.2 times, compared to non-encapsulated cardiac fibroblasts, mesenchymal stem cells, and ES cells. This reduction should minimize inflammatory and immune responses induced damage to the cells implanted in vivo becasue IgG binding is an important first step of the undesired host responses. Therefore, the ACA microcapsule with selective shell permeability should be of importance to advance the emerging cell-based medicine. PMID:23505611

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

    PubMed

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

    2016-09-01

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

  16. Enzymatically triggered peptide hydrogels for 3D cell encapsulation and culture.

    PubMed

    Szkolar, Laura; Guilbaud, Jean-Baptiste; Miller, Aline F; Gough, Julie E; Saiani, Alberto

    2014-07-01

    We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix. First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work. We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa). The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis. In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel. Human dermal fibroblasts were then used to exemplify the use of these materials. FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control. We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate. After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts. The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells.

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

    NASA Astrophysics Data System (ADS)

    Sasai, Yoshiki

    2013-05-01

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

  18. A cut cell method for the 3D simulation of Crookes radiometer

    SciTech Connect

    Dechriste, Guillaume; Mieussens, Luc

    2014-12-09

    Devices involved in engineering applications, such as vacuum pumps or MEMS, may be made of several moving parts. This raise the issue of the simulation of rarefied gas flow around moving boundaries. We propose a simple process, known as cut cell method, to treat the motion of a solid body in the framework of the deterministic solving of a kinetic equation. Up to our knowledge, this is the first time that this approach has been used for this kind of simulations. The method is illustrated by the 2D and 3D simulations of a Crookes radiometer.

  19. A cut cell method for the 3D simulation of Crookes radiometer

    NASA Astrophysics Data System (ADS)

    Dechriste, Guillaume; Mieussens, Luc

    2014-12-01

    Devices involved in engineering applications, such as vacuum pumps or MEMS, may be made of several moving parts. This raise the issue of the simulation of rarefied gas flow around moving boundaries. We propose a simple process, known as cut cell method, to treat the motion of a solid body in the framework of the deterministic solving of a kinetic equation. Up to our knowledge, this is the first time that this approach has been used for this kind of simulations. The method is illustrated by the 2D and 3D simulations of a Crookes radiometer.

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

    SciTech Connect

    Shao, Yan-Lin Faltinsen, Odd M.

    2014-10-01

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

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

    PubMed

    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.

  2. Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.

    PubMed

    Lenser, Thorsten; Weisshart, Klaus; Ulbricht, Tobias; Klement, Karolin; Hemmerich, Peter

    2010-01-01

    The three-dimensional (3D) architecture of the cell nucleus is determined not only by the presence of subnuclear domains, such as the nuclear envelope, chromosome territories, and nuclear bodies, but also by smaller domains which form in response to specific functions, such as RNA transcription, DNA replication, and DNA repair. Since both stable and dynamic structures contribute to nuclear morphology, it is important to study the biophysical principles of the formation of macromolecular assemblies within the nucleus. For this purpose, a variety of fluorescence fluctuation microscopy techniques can be applied. Here, we summarize our current knowledge on the 3D architecture of the mammalian cell nucleus and describe in detail how the assembly of functional nuclear protein complexes can be analyzed in living cells using fluorescence bleaching techniques, fluorescence correlation spectroscopy, raster image correlation spectroscopy, and mathematical modeling. In conclusion, the application of all these techniques in combination is a powerful tool to assess the full spectrum of nuclear protein dynamics and to understand the biophysical principles underlying nuclear structure and function.

  3. Wnt5a-mediating neurogenesis of human adipose tissue-derived stem cells in a 3D microfluidic cell culture system.

    PubMed

    Choi, Jeein; Kim, Sohyeun; Jung, Jinsun; Lim, Youngbin; Kang, Kyungsun; Park, Seungsu; Kang, Sookyung

    2011-10-01

    In stem cell biology, cell plasticity refers to the ability of stem cells to differentiate into a variety of cell lineages. Recently, cell plasticity has been used to refer to the ability of a given cell type to reversibly de-differentiate, re-differentiate, or transdifferentiate in response to specific stimuli. These processes are regulated by multiple intracellular and extracellular growth and differentiation factors, including low oxygen. Our recent study showed that 3D microfluidic cell culture induces activation of the Wnt5A/β-catenin signaling pathway in hATSCs (human Adipose Tissue-derived Stem Cells). This resulted in self renewal and transdifferentiation of hATSCs into neurons. To improve neurogenic potency of hATSCs in response to low oxygen and other unknown physical factors, we developed a gel-free 3D microfluidic cell culture system (3D-μFCCS). The functional structure was developed for the immobilization of 3D multi-cellular aggregates in a microfluidic channel without the use of a matrix on the chip. Growth of hATSCs neurosphere grown on a chip was higher than the growth of control cells grown in a culture dish. Induction of differentiation in the Chip system resulted in a significant increase in the induction of neuronal-like cell structures and the presentation of TuJ or NF160 positive long neuritis compared to control cells after active migration from the center of the microfluidic channel layer to the outside of the microfluidic channel layer. We also observed that the chip neurogenesis system induced a significantly higher level of GABA secreting neurons and, in addition, almost 60% of cells were GABA + cells. Finally, we observed that 1 month of after the transplantation of each cell type in a mouse SCI lesion, chip cultured and neuronal differentiated hATSCs exhibited the ability to effectively transdifferentiate into NF160 + motor neurons at a high ratio. Interestingly, our CHIP/PCR analysis revealed that HIF1α-induced hATSCs neurogenesis

  4. Fourier-Space Nonlinear Rayleigh-Taylor Growth Measurements of 3D Laser-Imprinted Modulations in Planar Targets

    SciTech Connect

    Smalyuk, V.A.; Sadot, O.; Delettrez, J.A.; Meyerhofer, D.D.; Regan, S.P.; Sangster, T.C.

    2005-12-05

    Nonlinear growth of 3-D broadband nonuniformities was measured near saturation levels using x-ray radiography in planar foils accelerated by laser light. The initial target modulations were seeded by laser nonuniformities and later amplified during acceleration by Rayleigh-Taylor instability. The nonlinear saturation velocities are measured for the first time and are found to be in excellent agreement with Haan predictions. The measured growth of long-wavelength modes is consistent with enhanced, nonlinear, long-wavelength generation in ablatively driven targets.

  5. Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time

    PubMed Central

    Baek, NamHuk; Seo, Ok Won; Kim, MinSung; Hulme, John; An, Seong Soo A

    2016-01-01

    Recently, increasing numbers of cell culture experiments with 3D spheroids presented better correlating results in vivo than traditional 2D cell culture systems. 3D spheroids could offer a simple and highly reproducible model that would exhibit many characteristics of natural tissue, such as the production of extracellular matrix. In this paper numerous cell lines were screened and selected depending on their ability to form and maintain a spherical shape. The effects of increasing concentrations of doxorubicin (DXR) on the integrity and viability of the selected spheroids were then measured at regular intervals and in real-time. In total 12 cell lines, adenocarcinomic alveolar basal epithelial (A549), muscle (C2C12), prostate (DU145), testis (F9), pituitary epithelial-like (GH3), cervical cancer (HeLa), HeLa contaminant (HEp2), embryo (NIH3T3), embryo (PA317), neuroblastoma (SH-SY5Y), osteosarcoma U2OS, and embryonic kidney cells (293T), were screened. Out of the 12, 8 cell lines, NIH3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U2OS formed regular spheroids and the effects of DXR on these structures were measured at regular intervals. Finally, 5 cell lines, A549, HeLa, SH-SY5Y, U2OS, and 293T, were selected for real-time monitoring and the effects of DXR treatment on their behavior were continuously recorded for 5 days. A potential correlation regarding the effects of DXR on spheroid viability and ATP production was measured on days 1, 3, and 5. Cytotoxicity of DXR seemed to occur after endocytosis, since the cellular activities and ATP productions were still viable after 1 day of the treatment in all spheroids, except SH-SY5Y. Both cellular activity and ATP production were halted 3 and 5 days from the start of the treatment in all spheroids. All cell lines maintained their spheroid shape, except SHSY-5, which behaved in an unpredictable manner when exposed to toxic concentrations of DXR. Cytotoxic effects of DXR towards SH-SY5Y seemed to cause degradation of

  6. Disease Modeling in Stem Cell-Derived 3D Organoid Systems.

    PubMed

    Dutta, Devanjali; Heo, Inha; Clevers, Hans

    2017-03-21

    Organoids are 3D in vitro culture systems derived from self-organizing stem cells. They can recapitulate the in vivo architecture, functionality, and genetic signature of original tissues. Thus, organoid technology has been rapidly applied to understanding stem cell biology, organogenesis, and various human pathologies. The recent development of human patient-derived organoids has enabled disease modeling with precision, highlighting their great potential in biomedical applications, translational medicine, and personalized therapy. In light of recent breakthroughs using organoids, it is only apt that we appreciate the advantages and shortcomings of this technology to exploit its full potential. We discuss recent advances in the application of organoids in studying cancer and hereditary diseases, as well as in the examination of host cell-microorganism interactions.

  7. Mesenchymal stem cell interactions with 3D ECM modules fabricated via multiphoton excited photochemistry.

    PubMed

    Su, Ping-Jung; Tran, Quyen A; Fong, Jimmy J; Eliceiri, Kevin W; Ogle, Brenda M; Campagnola, Paul J

    2012-09-10

    To understand complex micro/nanoscale ECM stem cell interactions, reproducible in vitro models are needed that can strictly recapitulate the relative content and spatial arrangement of native tissue. Additionally, whole ECM proteins are required to most accurately reflect native binding dynamics. To address this need, we use multiphoton excited photochemistry to create 3D whole protein constructs or "modules" to study how the ECM governs stem cell migration. The constructs were created from mixtures of BSA/laminin (LN) and BSA alone, whose comparison afforded studying how the migration dynamics are governed from the combination of morphological and ECM cues. We found that mesenchymal stem cells interacted for significantly longer durations with the BSA/LN constructs than pure BSA, pointing to the importance of binding cues of the LN. Critical to this work was the development of an automated system with feedback based on fluorescence imaging to provide quality control when synthesizing multiple identical constructs.

  8. Cancer Cell Migration within 3D Layer-By-Layer Microfabricated Photocrosslinked PEG Scaffolds with Tunable Stiffness

    PubMed Central

    Soman, Pranav; Kelber, Jonathan A.; Lee, Jin Woo; Wright, Tracy; Vecchio, Kenneth S.; Klemke, Richard L.; Chen, Shaochen

    2012-01-01

    Our current understanding of 3-dimensional (3D) cell migration is primarily based on results from fibrous scaffolds with randomly organized internal architecture. Manipulations that change the stiffness of these 3D scaffolds often alter other matrix parameters that can modulate cell motility independently or synergistically, making observations less predictive of how cells behave when migrating in 3D. In order to decouple microstructural influences and stiffness effects, we have designed and fabricated 3D polyethylene glycol (PEG) scaffolds that permit orthogonal tuning of both elastic moduli and microstructure. Scaffolds with log-pile architectures were used to compare the 3D migration properties of normal breast epithelial cells (HMLE) and Twist-transformed cells (HMLET). Our results indicate that the nature of cell migration is significantly impacted by the ability of cells to migrate in the third dimension. 2D ECM-coated PEG substrates revealed no statistically significant difference in cell migration between HMLE and HMLET cells among substrates of different stiffness. However, when cells were allowed to move along the third dimension, substantial differences were observed for cell displacement, velocity and path straightness parameters. Furthermore, these differences were sensitive to both substrate stiffness and the presence of the Twist oncogene. Importantly, these 3D modes of migration provide insight into the potential for oncogene-transformed cells to migrate within and colonize tissues of varying stiffness. PMID:22809641

  9. Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds with tunable stiffness.

    PubMed

    Soman, Pranav; Kelber, Jonathan A; Lee, Jin Woo; Wright, Tracy N; Vecchio, Kenneth S; Klemke, Richard L; Chen, Shaochen

    2012-10-01

    Our current understanding of 3-dimensional (3D) cell migration is primarily based on results from fibrous scaffolds with randomly organized internal architecture. Manipulations that change the stiffness of these 3D scaffolds often alter other matrix parameters that can modulate cell motility independently or synergistically, making observations less predictive of how cells behave when migrating in 3D. In order to decouple microstructural influences and stiffness effects, we have designed and fabricated 3D polyethylene glycol (PEG) scaffolds that permit orthogonal tuning of both elastic moduli and microstructure. Scaffolds with log-pile architectures were used to compare the 3D migration properties of normal breast epithelial cells (HMLE) and Twist-transformed cells (HMLET). Our results indicate that the nature of cell migration is significantly impacted by the ability of cells to migrate in the third dimension. 2D ECM-coated PEG substrates revealed no statistically significant difference in cell migration between HMLE and HMLET cells among substrates of different stiffness. However, when cells were allowed to move along the third dimension, substantial differences were observed for cell displacement, velocity and path straightness parameters. Furthermore, these differences were sensitive to both substrate stiffness and the presence of the Twist oncogene. Importantly, these 3D modes of migration provide insight into the potential for oncogene-transformed cells to migrate within and colonize tissues of varying stiffness.

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

    PubMed

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

    2014-08-07

    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.

  11. 3D Assessment of Mandibular Growth Based on Image Registration: A Feasibility Study in a Rabbit Model

    PubMed Central

    Kim, I.; Oliveira, M. E.; Duncan, W. J.; Cioffi, I.; Farella, M.

    2014-01-01

    Background. Our knowledge of mandibular growth mostly derives from cephalometric radiography, which has inherent limitations due to the two-dimensional (2D) nature of measurement. Objective. To assess 3D morphological changes occurring during growth in a rabbit mandible. Methods. Serial cone-beam computerised tomographic (CBCT) images were made of two New Zealand white rabbits, at baseline and eight weeks after surgical implantation of 1 mm diameter metallic spheres as fiducial markers. A third animal acted as an unoperated (no implant) control. CBCT images were segmented and registered in 3D (Implant Superimposition and Procrustes Method), and the remodelling pattern described used color maps. Registration accuracy was quantified by the maximal of the mean minimum distances and by the Hausdorff distance. Results. The mean error for image registration was 0.37 mm and never exceeded 1 mm. The implant-based superimposition showed most remodelling occurred at the mandibular ramus, with bone apposition posteriorly and vertical growth at the condyle. Conclusion. We propose a method to quantitatively describe bone remodelling in three dimensions, based on the use of bone implants as fiducial markers and CBCT as imaging modality. The method is feasible and represents a promising approach for experimental studies by comparing baseline growth patterns and testing the effects of growth-modification treatments. PMID:24527442

  12. The mouse retina in 3D: quantification of vascular growth and remodeling.

    PubMed

    Milde, Florian; Lauw, Stephanie; Koumoutsakos, Petros; Iruela-Arispe, M Luisa

    2013-12-01

    The mouse retina has become a prominent model for studying angiogenesis. The easy access and well-known developmental progression have significantly propelled our ability to examine and manipulate blood vessels in vivo. Nonetheless, most studies have restricted their evaluations to the superficial plexus (an upper vascular layer in contact with the vitreous). Here we present experimental data and quantification for the developmental progression of the full retina including the intermediate and deeper plexus that sprouts from the superficial layer. We analyze the origin and advancement of vertical sprouting and present the progression of vascular perfusion within the tissue. Furthermore, we introduce the use of Minkowsky functionals to quantify remodeling in the superficial and deeper plexus. The work expands information on the retina towards a 3D structure. This is of particular interest, as recent data have demonstrated differential effects of gene deletion on the upper and deeper plexus, highlighting the concept of distinct operational pathways during sprouting angiogenesis.

  13. Review on Chalcogenide 3D Nano-structured Crystals: Synthesis and Growth Mechanism.

    PubMed

    Qiu, Qi

    2015-01-01

    Three dimensional (3D) nano-structured crystals have received extensive attention for their superior properties over zero dimensional (0D), one dimensional (1D), or two dimensional (2D) nanomaterials in many areas. This review is generalized for the group of chalcogenide nanoflowers (NFs) by the synthetic techniques, such as solvothermal, wet chemical, sol-gel, surface oxidation, microwave, coating, electrochemical, and several other methods. The formation mechanism was also described for the purpose of opening up new food for thoughts to bring up new functionality of materials by tuning the morphology of crystals. The pH value or the template plays fundamental role in forming the nano-flowered structure. Moreover, the correlations between the surface area (SA), contact angle (CA), and the NFs are also discussed within the context. Here, we also discussed some patents relevant to the topic.

  14. Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes.

    PubMed

    Uz, Metin; Büyüköz, Melda; Sharma, Anup D; Sakaguchi, Donald S; Altinkaya, Sacide Alsoy; Mallapragada, Surya K

    2017-02-16

    In this study, gelatin-based 3D conduits with three different microstructures (nanofibrous, macroporous and ladder-like) were fabricated for the first time via combined molding and thermally induced phase separation (TIPS) technique for peripheral nerve regeneration. The effects of conduit microstructure and mechanical properties on the transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs) into Schwann cell (SC) like phenotypes were examined to help facilitate neuroregeneration and understand material-cell interfaces. Results indicated that 3D macroporous and ladder-like structures enhanced MSC attachment, proliferation and spreading, creating interconnected cellular networks with large numbers of viable cells compared to nanofibrous and 2D-tissue culture plate counterparts. 3D-ladder-like conduit structure with complex modulus of ∼0.4×10(6)Pa and pore size of ∼150μm provided the most favorable microenvironment for MSC transdifferentiation leading to ∼85% immunolabeling of all SC markers. On the other hand, the macroporous conduits with complex modulus of ∼4×10(6)Pa and pore size of ∼100μm showed slightly lower (∼65% for p75, ∼75% for S100 and ∼85% for S100β markers) immunolabeling. Transdifferentiated MSCs within 3D-ladder-like conduits secreted significant amounts (∼2.5pg/mL NGF and ∼0.7pg/mL GDNF per cell) of neurotrophic factors, while MSCs in macroporous conduits released slightly lower (∼1.5pg/mL NGF and 0.7pg/mL GDNF per cell) levels. PC12 cells displayed enhanced neurite outgrowth in media conditioned by conduits with transdifferentiated MSCs. Overall, conduits with macroporous and ladder-like 3D structures are promising platforms in transdifferentiation of MSCs for neuroregeneration and should be further tested in vivo.

  15. A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications.

    PubMed

    Jakubikova, Jana; Cholujova, Danka; Hideshima, Teru; Gronesova, Paulina; Soltysova, Andrea; Harada, Takeshi; Joo, Jungnam; Kong, Sun-Young; Szalat, Raphael E; Richardson, Paul G; Munshi, Nikhil C; Dorfman, David M; Anderson, Kenneth C

    2016-11-22

    Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM.

  16. A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications

    PubMed Central

    Jakubikova, Jana; Cholujova, Danka; Hideshima, Teru; Gronesova, Paulina; Soltysova, Andrea; Harada, Takeshi; Joo, Jungnam; Kong, Sun-Young; Szalat, Raphael E.; Richardson, Paul G.; Munshi, Nikhil C.; Dorfman, David M.; Anderson, Kenneth C.

    2016-01-01

    Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM. PMID:27764795

  17. Focal adhesion kinase activity is required for actomyosin contractility-based invasion of cells into dense 3D matrices

    PubMed Central

    Mierke, Claudia T.; Fischer, Tony; Puder, Stefanie; Kunschmann, Tom; Soetje, Birga; Ziegler, Wolfgang H.

    2017-01-01

    The focal adhesion kinase (FAK) regulates the dynamics of integrin-based cell adhesions important for motility. FAK’s activity regulation is involved in stress-sensing and focal-adhesion turnover. The effect of FAK on 3D migration and cellular mechanics is unclear. We analyzed FAK knock-out mouse embryonic fibroblasts and cells expressing a kinase-dead FAK mutant, R454-FAK, in comparison to FAK wild-type cells. FAK knock-out and FAKR454/R454 cells invade dense 3D matrices less efficiently. These results are supported by FAK knock-down in wild-type fibroblasts and MDA-MB-231 human breast cancer cells showing reduced invasiveness. Pharmacological interventions indicate that in 3D matrices, cells deficient in FAK or kinase-activity behave similarly to wild-type cells treated with inhibitors of Src-activity or actomyosin-contractility. Using magnetic tweezers experiments, FAKR454/R454 cells are shown to be softer and exhibit impaired adhesion to fibronectin and collagen, which is consistent with their reduced 3D invasiveness. In line with this, FAKR454/R454 cells cannot contract the matrix in contrast to FAK wild-type cells. Finally, our findings demonstrate that active FAK facilitates 3D matrix invasion through increased cellular stiffness and transmission of actomyosin-dependent contractile force in dense 3D extracellular matrices. PMID:28202937

  18. Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells.

    PubMed

    Vidyasekar, Prasanna; Shyamsunder, Pavithra; Sahoo, Sanjeeb Kumar; Verma, Rama Shanker

    2016-02-01

    3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or "mesoid bodies" (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Segmentation of 3D cell membrane images by PDE methods and its applications.

    PubMed

    Mikula, K; Peyriéras, N; Remešíková, M; Stašová, O

    2011-06-01

    We present a set of techniques that enable us to segment objects from 3D cell membrane images. Particularly, we propose methods for detection of approximate cell nuclei centers, extraction of the inner cell boundaries, the surface of the organism and the intercellular borders--the so called intercellular skeleton. All methods are based on numerical solution of partial differential equations. The center detection problem is represented by a level set equation for advective motion in normal direction with curvature term. In case of the inner cell boundaries and the global surface, we use the generalized subjective surface model. The intercellular borders are segmented by the advective level set equation where the velocity field is given by the gradient of the signed distance function to the segmented inner cell boundaries. The distance function is computed by solving the time relaxed eikonal equation. We describe the mathematical models, explain their numerical approximation and finally we present various possible practical applications on the images of zebrafish embryogenesis--computation of important quantitative characteristics, evaluation of the cell shape, detection of cell divisions and others.

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

    PubMed

    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

  2. Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

    PubMed

    Kamei, Ken-Ichiro; Koyama, Yoshie; Tokunaga, Yumie; Mashimo, Yasumasa; Yoshioka, Momoko; Fockenberg, Christopher; Mosbergen, Rowland; Korn, Othmar; Wells, Christine; Chen, Yong

    2016-11-01

    Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

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

    PubMed

    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.

  4. 3D-Mössbauer spectroscopic microscope for mc-Si solar cell evaluation

    NASA Astrophysics Data System (ADS)

    Ino, Y.; Soejima, H.; Hayakawa, K.; Yukihira, K.; Tanaka, K.; Fujita, H.; Watanabe, T.; Ogai, K.; Moriguchi, K.; Harada, Y.; Yoshida, Y.

    2016-12-01

    A 3D-Mössbauer Spectroscopic Microscope is developed to evaluate Fe impurities in multi-crystalline Si solar cells, which combines the Mössbauer spectroscopic microscope with a scanning electron microscope (SEM), an electron beam induced current (EBIC), an electron backscatter diffraction (EBSD), and an electron energy analyzer (HV-CSA). In addition, a new moving-coil-actuator with a liner encoder of 100 nm-resolution is incorporated for the operations with both a constant velocity and a constant acceleration mode successfully with the same precision as that obtained by the conventional transducers. Furthermore, a new multi-capillary X-ray lens is designed to achieve a γ-ray spot size less than 100 μm in diameter. The new microscope provides us to investigate the space correlation between Fe impurities and the lattice defects such as grain boundaries in multi-crystalline Si solar cells.

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

    SciTech Connect

    Rainey, E. S. G.; Kavner, A.; Hernlund, J. W.

    2013-11-28

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

  6. Cell culture and characterization of cross-linked poly(vinyl alcohol)-g-starch 3D scaffold for tissue engineering.

    PubMed

    Hsieh, Wen-Chuan; Liau, Jiun-Jia

    2013-10-15

    The research goal of this experiment is chemically to cross-link poly(vinyl alcohol) (PVA) and starch to form a 3D scaffold that is effective water absorbent, has a stable structure, and supports cell growth. PVA and starch can be chemically cross-linked to form a PVA-g-starch 3D scaffold polymer, as observed by Fourier transform infrared spectroscopy (FTIR), with an absorbency of up to 800%. Tensile testing reveals that, as the amount of starch increases, the strength of the 3D scaffold strength reaches 4×10(-2) MPa. Scanning electron microscope (SEM) observations of the material reveal that the 3D scaffold is highly porous formed using a homogenizer at 500 rpm. In an enzymatic degradation, the 3D scaffold was degraded by various enzymes at a rate of up to approximately 30-60% in 28 days. In vitro tests revealed that cells proliferate and grow in the 3D scaffold material. Energy dispersive spectrometer (EDS) analysis further verified that the bio-compatibility of this scaffold.

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

    PubMed

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

    2013-11-07

    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.

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

    PubMed

    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).

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

    PubMed

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

    2015-08-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  12. Computational models of hair cell bundle mechanics: III. 3-D utricular bundles.

    PubMed

    Silber, Joe; Cotton, John; Nam, Jong-Hoon; Peterson, Ellengene H; Grant, Wally

    2004-11-01

    Six utricular hair bundles from a red-eared turtle are modeled using 3-D finite element analysis. The mechanical model includes shear deformable stereocilia, realignment of all forces during force load increments, and tip and lateral link inter-stereocilia connections. Results show that there are two distinct bundle types that can be separated by mechanical bundle stiffness. The more compliant group has fewer total stereocilia and short stereocilia relative to kinocilium height; these cells are located in the medial and lateral extrastriola. The stiff group are located in the striola. They have more stereocilia and long stereocilia relative to kinocilia heights. Tip link tensions show parallel behavior in peripheral columns of the bundle and serial behavior in central columns when the tip link modulus is near or above that of collagen (1x10(9) N/m(2)). This analysis shows that lumped parameter models of single stereocilia columns can show some aspects of bundle mechanics; however, a distributed, 3-D model is needed to explore overall bundle behavior.

  13. 2D/3D perovskite hybrids as moisture-tolerant and efficient light absorbers for solar cells.

    PubMed

    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.

  14. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity

    PubMed Central

    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

  15. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    PubMed

    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.

  16. 3D Culture of MIN-6 Cells on Decellularized Pancreatic Scaffold: In Vitro and In Vivo Study

    PubMed Central

    Wu, Di; Wan, Jian; Huang, Yan; Guo, Yibing; Xu, Tianxin; Zhu, Mingyan; Fan, Xiangjun; Zhu, Shajun; Ling, Changchun; Li, Xiaohong; Lu, Jingjing; Zhu, Hui; Zhou, Pengcheng; Lu, Yuhua; Wang, Zhiwei

    2015-01-01

    Type 1 diabetes is an autoimmune disease which is due to the lack of β cells. The ideal therapy to cure the disease is pancreas transplantation, but its application is confined to a limited number of people due to the shortage of organ and the need for life-long immunosuppression. Regenerative medicine methods such as a tissue engineered pancreas seem to provide a useful method. In order to construct a microenvironment similar to the native pancreas that is suitable for not only cell growth but also cellular function exertion, a decellularized mouse pancreas was used as a natural 3D scaffold in this experiment. MIN-6 β cells were planted in the bioscaffold. The cell engraftment was verified by HE staining and SEM. Immunostaining procedures were performed to confirm the normal function of the engrafted cells. qRT-PCR demonstrated that insulin gene expression of the recellularized pancreas was upregulated compared with conventional plate-cultured cells. In vivo experiment was also accomplished to further evaluate the function of the recellularized bioscaffold and the result was inspiring. And beyond doubt this will bring new hope for type 1 diabetic patients. PMID:26688810

  17. An interface reconstruction method based on an analytical formula for 3D arbitrary convex cells

    DOE PAGES

    Diot, Steven; François, Marianne M.

    2015-10-22

    In this study, we are interested in an interface reconstruction method for 3D arbitrary convex cells that could be used in multi-material flow simulations for instance. We assume that the interface is represented by a plane whose normal vector is known and we focus on the volume-matching step that consists in finding the plane constant so that it splits the cell according to a given volume fraction. We follow the same approach as in the recent authors' publication for 2D arbitrary convex cells in planar and axisymmetrical geometries, namely we derive an analytical formula for the volume of the specificmore » prismatoids obtained when decomposing the cell using the planes that are parallel to the interface and passing through all the cell nodes. This formula is used to bracket the interface plane constant such that the volume-matching problem is rewritten in a single prismatoid in which the same formula is used to find the final solution. Finally, the proposed method is tested against an important number of reproducible configurations and shown to be at least five times faster.« less

  18. An interface reconstruction method based on an analytical formula for 3D arbitrary convex cells

    SciTech Connect

    Diot, Steven; François, Marianne M.

    2015-10-22

    In this study, we are interested in an interface reconstruction method for 3D arbitrary convex cells that could be used in multi-material flow simulations for instance. We assume that the interface is represented by a plane whose normal vector is known and we focus on the volume-matching step that consists in finding the plane constant so that it splits the cell according to a given volume fraction. We follow the same approach as in the recent authors' publication for 2D arbitrary convex cells in planar and axisymmetrical geometries, namely we derive an analytical formula for the volume of the specific prismatoids obtained when decomposing the cell using the planes that are parallel to the interface and passing through all the cell nodes. This formula is used to bracket the interface plane constant such that the volume-matching problem is rewritten in a single prismatoid in which the same formula is used to find the final solution. Finally, the proposed method is tested against an important number of reproducible configurations and shown to be at least five times faster.

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

    PubMed Central

    Annibale, Paolo; Gratton, Enrico

    2015-01-01

    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. PMID:25788248

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

    PubMed

    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.

  1. 3D structures of individual mammalian genomes studied by single-cell Hi-C.

    PubMed

    Stevens, Tim J; Lando, David; Basu, Srinjan; Atkinson, Liam P; Cao, Yang; Lee, Steven F; Leeb, Martin; Wohlfahrt, Kai J; Boucher, Wayne; O'Shaughnessy-Kirwan, Aoife; Cramard, Julie; Faure, Andre J; Ralser, Meryem; Blanco, Enrique; Morey, Lluis; Sansó, Miriam; Palayret, Matthieu G S; Lehner, Ben; Di Croce, Luciano; Wutz, Anton; Hendrich, Brian; Klenerman, Dave; Laue, Ernest D

    2017-04-06

    The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

  2. 3D differentiation of neural stem cells in macroporous photopolymerizable hydrogel scaffolds.

    PubMed

    Li, Hang; Wijekoon, Asanka; Leipzig, Nic D

    2012-01-01

    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.

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

    PubMed

    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.

  4. 3D Differentiation of Neural Stem Cells in Macroporous Photopolymerizable Hydrogel Scaffolds

    PubMed Central

    Li, Hang; Wijekoon, Asanka; Leipzig, Nic D.

    2012-01-01

    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 µm2, 10 wt%, 6330±1160 µm2, 20 wt%, 7600±1550 µm2) compared with controls (0 wt%, 3150±220 µm2). 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. PMID:23144988

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    SciTech Connect

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

    2009-06-19

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

  8. Transition from Ignition to Flame Growth under External Radiation in Three Dimensions (TIGER-3D)

    NASA Technical Reports Server (NTRS)

    Kashiwagi, Takashi; Nakamura, Yuji; Olson, Sandra L.; Mell, William

    2004-01-01

    This study focuses on localized ignition by external radiant flux and subsequent flame growth over thin polymeric materials (plastic and paper) in microgravity. Two transition stages were observed. The first transition stage covers the period from the onset of ignition to the formation of stabilized flame near the ignited area. This is followed by the second transition of the flame growth stage from the initial stabilized flame to sustained fire growth away from the ignited area. For the first stage, ignition experiments of thin PMMA sheets were conducted using a CO2 laser as an external source in the 10 s drop tower. The results of front side surface ignition and of backside surface ignition were observed. The effects of imposed flow velocity, sample thickness, and ambient oxygen concentration on ignition are obtained. Numerical study was conducted to investigate to understand and predict ignition behavior observed in the experiments. For the second stage, numerical study is being conducted to describe the effects of gravity on heat release rate of a PMMA sheet. The gravity level was varied from zero to normal gravity. The preliminary results show that the maximum heat release occurs at around 0.02 g.

  9. 3-D simulations to investigate initial condition effects on the growth of Rayleigh-Taylor mixing

    SciTech Connect

    Andrews, Malcolm J

    2008-01-01

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

  10. Measuring dynamic cell-material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels.

    PubMed

    Schultz, Kelly M; Kyburz, Kyle A; Anseth, Kristi S

    2015-07-21

    Biomaterials that mimic aspects of the extracellular matrix by presenting a 3D microenvironment that cells can locally degrade and remodel are finding increased applications as wound-healing matrices, tissue engineering scaffolds, and even substrates for stem cell expansion. In vivo, cells do not simply reside in a static microenvironment, but instead, they dynamically reengineer their surroundings. For example, cells secrete proteases that degrade extracellular components, attach to the matrix through adhesive sites, and can exert traction forces on the local matrix, causing its spatial reorganization. Although biomaterials scaffolds provide initially well-defined microenvironments for 3D culture of cells, less is known about the changes that occur over time, especially local matrix remodeling that can play an integral role in directing cell behavior. Here, we use microrheology as a quantitative tool to characterize dynamic cellular remodeling of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels that degrade in response to cell-secreted matrix metalloproteinases (MMPs). This technique allows measurement of spatial changes in material properties during migration of encapsulated cells and has a sensitivity that identifies regions where cells simply adhere to the matrix, as well as the extent of local cell remodeling of the material through MMP-mediated degradation. Collectively, these microrheological measurements provide insight into microscopic, cellular manipulation of the pericellular region that gives rise to macroscopic tracks created in scaffolds by migrating cells. This quantitative and predictable information should benefit the design of improved biomaterial scaffolds for medically relevant applications.