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Scaffold coated with long-sugar molecules enhances stem-cell cultures

Stem cells have the unique ability to turn into any type of human cell, opening up all sorts of therapeutic possibilities for some of the world’s incurable diseases and conditions. The problem facing scientists is how to encourage stem cells to turn into the particular type of cell required to treat a specific disease.

 

 

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Chapter 7 - Methods for Analysis of Apical Lumen Trafficking Using Micropatterned 3D Systems

Chapter 7 - Methods for Analysis of Apical Lumen Trafficking Using Micropatterned 3D Systems | Micropatterns | Scoop.it

Epithelial organs are made of interconnected branched networks of tubules, with a central lumen lined by a monolayer of epithelial cells. Certain epithelial cell lines can be converted into organotypic cultures by the addition of extracellular matrix components. When cultured in these conditions, epithelial cells reorient the axis of polarity, reorganize the membrane surfaces, and transport apical proteins to form the lumen in a process that recapitulates essential aspects of de novo apical membrane formation during epithelial organ morphogenesis. Micropatterns are a simple technique that allows cell culture in a controlled adhesive environment with extremely high precision, close to the nanometer scale. We have recently developed a method to culture MDCK cysts on micropatterns of different sizes and composition. Using this method we found that changes in micropattern shape and size can be used to modify cell contractility to understand its contribution to apical membrane formation. When imaging cysts on micropatterns the main advantage is that apical-directed vesicle trafficking is visualized in the x–y plane, which presents higher resolution on confocal microscopes. Thus, the use of micropatterns is an efficient setup to analyze polarized secretion with unprecedented higher resolution in both time and space.

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Two-Photon Polymerization of Sub-Micrometric Patterned Surfaces: Investigation of Cell-Substrate Interactions and Improved Differentiation of Neuron-Like Cells

Two-Photon Polymerization of Sub-Micrometric Patterned Surfaces: Investigation of Cell-Substrate Interactions and Improved Differentiation of Neuron-Like Cells | Micropatterns | Scoop.it

Direct Laser Writing (DLW) is an innovative tool that allows the photo-fabrication of high resolution 3D structures, that can be successfully exploited for the study of the physical interactions between cells and substrates. In this work, we focused our attention on the topographical effects of sub-micrometric patterned surfaces fabricated via DLW on neuronal cell behavior. In particular, we designed, prepared, and characterized substrates based on aligned ridges for the promotion of axonal outgrowth and guidance. We demonstrated that both rat PC12 neuron-like cells and human SH-SY5Y derived neurons differentiate on parallel 2.5 µm spaced sub-micrometric ridges, being characterized by strongly aligned and significantly longer neurites with respect to those differentiated on flat control substrates, or on more spaced (5 and 10 µm) ridges. Furthermore, we detected an increased molecular differentiation toward neurons of the SH-SY5Y cells when grown on the sub-micrometric patterned. Finally, we observed that the axons can exert forces able of bending the ridges, and we indirectly estimated the order of magnitude of these forces thanks to scanning probe techniques. Collectively, we showed as sub-micrometric structures fabricated by DLW can be used as a useful tool for the study of the axon mechanobiology.

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Epithelial bridges maintain tissue integrity during collective cell migration

Epithelial bridges maintain tissue integrity during collective cell migration | Micropatterns | Scoop.it

The ability of skin to act as a barrier is primarily determined by the efficiency of skin cells to maintain and restore its continuity and integrity. In fact, during wound healing keratinocytes migrate collectively to maintain their cohesion despite heterogeneities in the extracellular matrix. Here, we show that monolayers of human keratinocytes migrating along functionalized micropatterned surfaces comprising alternating strips of extracellular matrix (fibronectin) and non-adherent polymer form suspended multicellular bridges over the non-adherent areas. The bridges are held together by intercellular adhesion and are subjected to considerable tension, as indicated by the presence of prominent actin bundles. We also show that a model based on force propagation through an elastic material reproduces the main features of bridge maintenance and tension distribution. Our findings suggest that multicellular bridges maintain tissue integrity during wound healing when cell–substrate interactions are weak and may prove helpful in the design of artificial scaffolds for skin regeneration.

 

 

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A review of microfabrication and hydrogel engineering for micro-organs on chips

A review of microfabrication and hydrogel engineering for micro-organs on chips | Micropatterns | Scoop.it

This review highlights recent trends towards the development of in vitro multicellular systems with definite architectures, or “organs on chips”. First, the chemical composition and mechanical properties of the scaffold have to be consistent with the anatomical environment in vivo. In this perspective, the flourishing interest in hydrogels as cellular substrates has highlighted the main parameters directing cell differentiation that need to be recapitulated in artificial matrix. Another scaffold requirement is to act as a template to guide tissue morphogenesis. Therefore specific microfabrication techniques are required to spatially pattern the environment at microscale. 2D patterning is particularly efficient for organizing planar polarized cell types such as endothelial cells or neurons. However, most organs are characterized by specific sub units organized in three dimensions at the cellular level. The reproduction of such 3D patterns in vitro is necessary for cells to fully differentiate, assemble and coordinate to form a coherent micro-tissue. These physiological microstructures are often integrated in microfluidic devices whose controlled environments provide the cell culture with more life-like conditions than traditional cell culture methods. Such systems have a wide range of applications, for fundamental research, as tools to accelerate drug development and testing, and finally, for regenerative medicine.

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Influence of the pattern size of micropatterned scaffolds on cell morphology, proliferation, migration and F-actin expression

Influence of the pattern size of micropatterned scaffolds on cell morphology, proliferation, migration and F-actin expression | Micropatterns | Scoop.it

To determine how the three-dimensional (3D) shape of scaffolds influences cell functions, 3D micropatterned scaffolds of various sizes were fabricated on a silicon substrate. The micropatterns were equilateral triangular pores with 3–20 μm long sides, and all had the same pore ratio (total pore area per unit area) and depth. The patterns only differed in terms of their 2D size. Such scaffolds have not been previously generated, and thus the effects of pattern size on cell functions have not been addressed. NIH-3T3 cells were cultured on these micropatterned scaffolds, and their morphology, proliferation rate, migration rate, and level of F-actin expression were assessed. Cells became more rounded and F-actin expression decreased as the pattern size of the scaffold decreased. Relationships were also demonstrated between pattern size and cell proliferation and migration. These results suggest that the pattern size of 3D micropatterned scaffolds affects the level of mechanical stress that cells experience, and thereby influences F-actin expression, cell morphology, cell proliferation and cell migration.

 

 

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Surface-printed microdot array chips for the quantification of axonal collateral branching of a single neuron in vitro

Surface-printed microdot array chips for the quantification of axonal collateral branching of a single neuron in vitro | Micropatterns | Scoop.it

Precise and quantitative control of extracellular signalling cues using surface-engineered chips has facilitated various neurobiological assays in vitro. Although the formation of axon collateral branches is important for the establishment and refinement of the neuronal connections during the development and regeneration, surface designs for controlling branch phenotypes have been rarely proposed. In this work, we fabricated a surface-printed microdot array for controlling axon branch formation. Following the culture of hippocampal neurons on a 5 μm dot array patterned by micro-contact printing of poly-D-lysine, we found that most axon collateral branches were initiated from axonal regions on a microdot and terminated on neighboring dots. In addition, the length of branches increased as the spacing between dots increased. Surprisingly, other morphological features were not significantly different from the neurons cultured on a conventional unpatterned surface. Further investigation of this phenomenon indicated that the branch-forming machineries, such as actin patches, were focused on the dot. According to these investigations, we concluded that discontinuous adhesion spots given by dot arrays arranged the branching formation on the expectable location and direction. Therefore, microdot arrays will be applicable as the surface design parameter of bio-chip platforms to reduce branching complexity and quantize branching formation for the simple and easy assay in neurobiological studies.

 

 

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Specific rare cell capture using micro-patterned silicon nanowire platform

Specific rare cell capture using micro-patterned silicon nanowire platform | Micropatterns | Scoop.it

We report on the rapid and direct quantification of specific cell captures using a micro-patterned streptavidin (STR)-functionalized silicon nanowire (SiNW) platform, which was prepared by Ag-assisted wet chemical etching and a photo-lithography process. This platform operates by high-affinity cell capture rendered by the combination of antibody–epithelial cell surface-binding, biotin–streptavidin binding, and the topologically enhanced cell-substrate interaction on a 3-dimensional SiNWs array. In this work, we developed a micro-patterned nanowire platform, with which we were able to directly evaluate the performance enhancement due to nanotopography. An excellent capture efficiency of ~96.6±6.7%, which is the highest value achieved thus far for the targeting specific A549 cells on a selective area of patterned SiNWs, is demonstrated. Direct comparison between the nanowire region and the planar region on the same substrate indicates dramatically elevated cell-capture efficiency on nanotopological surface identical surface chemistry (<2% cell-capture efficiency). An excellent linear response was seen for quantifying captured A549 cells with respect to loaded cells. This study suggests that the micro-patterned STR-functionalized SiNWs platform provides additional advantage for detecting rare cells populations in a more quantitative and specific manner.

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The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part I (Migration)

The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part I (Migration) | Micropatterns | Scoop.it
Abstract: Initial cell-surface interactions are guided by the material properties of substrate topography. To examine if these interactions are also modulated by the presence of zinc, we seeded murine pre-osteoblasts (MC3T3-E1, subclone 4) on micropatterned polydimethylsiloxane (PDMS) containing wide (20 µm width, 30 µm pitch, 2 µm height) or narrow (2 µm width, 10 µm pitch, 2 µm height) ridges, with flat PDMS and tissue culture polystyrene (TC) as controls. Zinc concentration was adjusted to mimic deficient (0.23 µM), serum-level (3.6 µM), and zinc-rich (50 µM) conditions. Significant differences were observed in regard to cell morphology, motility, and contact guidance. We found that cells exhibited distinct anisotropic migration on the wide PDMS patterns under either zinc-deprived (0.23 µM) or serum-level zinc conditions (3.6 µM). However, this effect was absent in a zinc-rich environment (50 µM). These results suggest that the contact guidance of pre-osteoblasts may be partly influenced by trace metals in the microenvironment of the extracellular matrix.

 

 

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Material control of stem cell differentiation: challenges in nano-characterization

Material control of stem cell differentiation: challenges in nano-characterization | Micropatterns | Scoop.it

Recent experiments have revealed that stem cells respond to biophysical cues as well as numerous biochemical factors. Nanoscale properties at the cell–matrix interface that appear to affect adherent stem cells range from matrix elasticity to porosity-dependent matrix tethering and geometry of adhesive linkages. Some stem cells can also remodel their immediate environment to influence phenotype, but this depends on matrix-material properties such as covalent bonding and soft versus hard materials. Efforts to combine both matrix instructions and active cell feedback are required to properly direct stem cell behavior. Comparisons to tissues will be increasingly key and have begun to reveal remodeling of nuclear factors that influence epigenetics.

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Geometric control of vimentin intermediate filaments

Geometric control of vimentin intermediate filaments | Micropatterns | Scoop.it

Significant efforts have addressed the role of vimentin intermediate filaments (VIF) in cell motility, shape, adhesion and their connections to microfilaments (MF) and microtubules (MT). The present work uses micropatterned substrates to control the shapes of mouse fibroblasts and demonstrates that the cytoskeletal elements are dependent on each other and that unlike MF, VIF are globally controlled. For example, both square and circle shaped cells have a similar VIF distribution while MF distributions in these two shapes are quite different and depend on the curvature of the shape. Furthermore, in asymmetric and polarized shaped cells VIF avoid the sharp edges where MF are highly localized. Experiments with vimentin null mouse embryonic fibroblasts (MEFs) adherent to polarized (teardrop) and un-polarized (dumbbell) patterns show that the absence of VIF alters microtubule organization and perturbs cell polarity. The results of this study also demonstrate the utility of patterned substrates for quantitative studies of cytoskeleton organization in adherent cells.

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Physical Explanation of Coupled Cell-Cell Rotational Behavior and Interfacial Morphology: A Particle Dynamics Model

Physical Explanation of Coupled Cell-Cell Rotational Behavior and Interfacial Morphology: A Particle Dynamics Model | Micropatterns | Scoop.it

Previous studies have reported persistent rotational behavior between adherent cell-cell pairs cultured on micropatterned substrates, and this rotation is often accompanied by a sigmoidal deflection of the cell-cell interface. Interestingly, the cell-cell rotation runs in the opposite reference frame from what could be expected of single cell locomotion. Specifically, the rotation of the cell pair consists of each individual cell protruding from the inwardly regressive arm of the cell-cell interface, and retracting from the other outwardly protrusive arm. To this author’s knowledge, the cause of this elusive behavior has not yet been clarified. Here, we propose a physical model based on particle dynamics, accounting for actomyosin forcing, viscous dissipation, and cortical tension. The results show that a correlation in actomyosin force vectors leads to both persistent rotational behavior and interfacial deflection in a simulated cell cluster. Significantly, the model, without any artificial cues, spontaneously and consistently reproduces the same rotational reference frame as experimentally observed. Further analyses show that the interfacial deflection depends predominantly on cortical tension, whereas the cluster rotation depends predominantly on actomyosin forcing. Together, these results corroborate the hypothesis that both rotational and morphological phenomena are, in fact, physically coupled by an intracellular torque of a common origin.

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Nanofilm micropatterns for biomedical applications

Nanofilm micropatterns for biomedical applications | Micropatterns | Scoop.it

Novel multilayered nanofilm patterns are investigated by David Mills and his team from Louisiana Tech University as a basis for manufacturing engineered tissues, cellular arrays, biosensors and lab-on-chip devices.  The goal of these studies is to develop micropatterned surfaces either mimicking in vivo microenvironments that would allow the cells to grow and develop more naturally, or to alter cellular behavior in specific ways.  The scientists used primary canine chondrocytes (obtained from Cell Applications, Inc.) to evaluate 20 different nanofilm compositions.

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Neural circuits with long-distance axon tracts for determining functional connectivity

Neural circuits with long-distance axon tracts for determining functional connectivity | Micropatterns | Scoop.it

The cortical circuitry in the brain consists of structurally and functionally distinct neuronal assemblies with reciprocal axon connections. To generate cell culture-based systems that emulate axon tract systems of an in vivo neural network, we developed a living neural circuit consisting of compartmentalized neuronal populations connected by arrays of two millimeter-long axon tracts that are integrated on a planar multi-electrode array (MEA). The millimeter-scale node-to-node separation allows for pharmacological and electrophysiological manipulations to simultaneously target multiple neuronal populations. The results show controlled selectivity of dye absorption by neurons in different compartments. MEA-transmitted electrical stimulation of targeted neurons shows ∼46% increase of intracellular calcium levels with 20 Hz stimulation, but ∼22% decrease with 2k Hz stimulation. The unique feature of long distance axons promotes in vivo-like fasciculation. These axon tracts are determined to be inhibitory afferents by showing increased action potential firing of downstream node upon selective application of γ-aminobutyric acid (GABA) to the upstream node. Together, this model demonstrates integrated capabilities for assessing multiple endpoints including axon tract tracing, calcium influx, network architecture and activities. This system can be used as a multi-functional platform for studying axon tract-associated CNS disorders in vitro, such as diffuse axonal injury after brain trauma.

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Studying Intracellular Trafficking Pathways with Probabilistic Density Maps

Studying Intracellular Trafficking Pathways with Probabilistic Density Maps | Micropatterns | Scoop.it

The compartmentalization of cellular functions in complex membranous organelles is a key feature of eukaryotic cells. To cope with the enormous complexity of trafficking pathways that connect these compartments, new approaches need to be considered and introduced into the field of cell biology. We exploit the advantages of the “micropatterning technique,” which is to bring cells to adopt a highly reproducible shape, and probabilistic density mapping, which quantifies spatial organization of trafficking compartments, to study regulatory mechanisms of intracellular trafficking. Here, we provide a protocol to analyze and quantify alterations in trafficking compartments upon cellular manipulation. We demonstrate how this approach can be employed to study the regulation of Rab6-labeled transport carriers by the cytoskeleton.

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Reprogramming hMSCs morphology with silicon/porous silicon geometric micro-patterns

Reprogramming hMSCs morphology with silicon/porous silicon geometric micro-patterns | Micropatterns | Scoop.it

Geometric micro-patterned surfaces of silicon combined with porous silicon (Si/PSi) have been manufactured to study the behaviour of human Mesenchymal Stem Cells (hMSCs). These micro-patterns consist of regular silicon hexagons surrounded by spaced columns of silicon equilateral triangles separated by PSi. The results show that, at an early culture stage, the hMSCs resemble quiescent cells on the central hexagons with centered nuclei and actin/β-catenin and a microtubules network denoting cell adhesion. After 2 days, hMSCs adapted their morphology and cytoskeleton proteins from cell-cell dominant interactions at the center of the hexagonal surface. This was followed by an intermediate zone with some external actin fibres/β-catenin interactions and an outer zone where the dominant interactions are cell-silicon. Cells move into silicon columns to divide, migrate and communicate. Furthermore, results show that Runx2 and vitamin D receptors, both specific transcription factors for skeleton-derived cells, are expressed in cells grown on micropatterned silicon under all observed circumstances. On the other hand, non-phenotypic alterations are under cell growth and migration on Si/PSi substrates. The former consideration strongly supports the use of micro-patterned silicon surfaces to address pending questions about the mechanisms of human bone biogenesis/pathogenesis and the study of bone scaffolds.

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Miniaturized pre-clinical cancer models as research and diagnostic tools

Miniaturized pre-clinical cancer models as research and diagnostic tools | Micropatterns | Scoop.it

Cancer is one of the most common causes of death worldwide. Consequently, important resources are directed towards bettering treatments and outcomes. Cancer is difficult to treat due to its heterogeneity, plasticity and frequent drug resistance. New treatment strategies should strive for personalized approaches. These should target neoplastic and/or activated microenvironmental heterogeneity and plasticity without triggering resistance and spare host cells. In this review, the putative use of increasingly physiologically relevant microfabricated cell-culturing systems intended for drug development is discussed. There are two main reasons for the use of miniaturized systems. First, scaling down model size allows for high control of microenvironmental cues enabling more predictive outcomes. Second, miniaturization reduces reagent consumption, thus facilitating combinatorial approaches with little effort and enables the application of scarce materials, such as patient-derived samples. This review aims to give an overview of the state-of-the-art of such systems while predicting their application in cancer drug development.

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Cardiomyocyte sensor responsive to changes in physical and chemical environments

Cardiomyocyte sensor responsive to changes in physical and chemical environments | Micropatterns | Scoop.it

Conventional cardiac physiology experiments investigate in vitro beat frequency using cells isolated from adult or neonatal rat hearts. In this study, we show that various cantilever shapes and drug treatments alter cardiomyocyte contraction force in vitro. Four types of cantilevers were used to compare the contractile forces: flat, peg patterned, grooved, and peg and grooved. Contraction force was represented as bending deflection of the cantilever end. The deflections of the flat, peg patterned, grooved, and peg and grooved cantilevers were 24.2 nN, 41.6 nN, 121 nN, and 134.2 nN, respectively. We quantified the effect of drug treatments on cardiomyocyte contractile forces on the grooved cantilever using Digoxin, Isoproterenol, and BayK8644, all of which increase contractile force, and Verapamil, which decreases contractile force. The cardiomyocyte contractile force without drugs decreased 8 days after culture initiation. Thus, we applied Digoxin, Isoproterenol, and BayK8644 at day 8, and Verapamil at day 5. Digoxin, Isoproterenol, and BayK8644 increased the cardiomyocyte contractile forces by 19.31%, 9.75%, and 23.81%, respectively. Verapamil decreased the contraction force by 48.06%. In summary, contraction force changes in response to adhesion surface topology and various types of drug treatments. We observed these changes by monitoring cell alignment, adhesion, morphology, and bending displacement with cantilever sensors.

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Mechanical properties of the plasma membrane of micropatterned cells incubated with mono or polyunsaturated fatty acids

Some mammalian cells show striking differences in the acyl chain composition of their membrane phospholipids. In most cases, the majority of phospholipids bear one saturated and one monounsaturated acyl chains at positions 1 and 2 or the glycerol, respectively. However, some cells and notably neurons contain large amounts of phospholipids with a polyunsaturated fatty acyl chain, generally at position 2. The aim of this project is to compare the impact of the phospholipid polyunsaturation vs monounsaturation on the mechanical and functional properties of the plasma membrane. For this, Immortalized Retinal Pigmental Epithelial Cells (RPE1) were cultured with various BSA-fatty acid complexes. Gas chromatography and mass spectrometry show that these cells incorporate most fatty acids in their phospholipids, preferentially in phosphatidylcholine. Notably, docosahexaenic acid (C22:6) levels in phospholipids increase from trace amounts to 5-10 mol % after a few hours. We then compared the plasma membrane elastic properties during incubation with C18:1 or C22:6. The cells were cultured on L-shaped fibronectin micropatterns and a membrane tubule was pulled from the cell hypothenus using optical tweezers. C22:6 caused a gradual 2-fold decrease within an hour of both the pulling force (F) and the tube radius (R) whereas C18:1 caused only modest changes. From these measurements, we conclude that the bending modulus (which depends on the FxR product) strongly decreases with phospholipid polyunsaturation, whereas the tension of the plasma membrane (which depends on the F/R ratio) is mostly unaffected. These results are in good agreement with studies performed on model phospholipid membranes (Biophys J 79, 328-39). We currently investigate how phospholipid insaturation affects the ability of specialized protein machineries involved in transport vesicle formation in RPE1 cells, focusing on the impact of C22:6 compared to C18:1 on the rates of transferrin and EGF endocytosis.

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Micropatterning of ECM Proteins on Glass Substrates to Regulate Cell Attachment and Proliferation

Background

Micropatterning is becoming a powerful tool for studying cells in vitro. This method not only uses very small amount of material but also mimic the microenvironment structure present in living tissues better than flask culturing techniques. In previous studies using micropatterning of extracellular matrix proteins on glass surfaces, the rate of protein detachment from the surface was so high that the proteins and the cultivated cells detached after 3 three days of cell seeding.

Methods

Here we optimized the glass surface modification method to fulfill the requirement of most in vitro studies.

Results

In our study we showed that the optimum time for glass surface modification reaction is 1.5 hr, and the cells could be tracked in vitro for over 15 days after cell seeding which is enough for the most in vitro studies. As a model, we cultivated HEK 293T and HepG2 cells on the collagen micro-patterns and showed that they have normal growth and morphology on these micropatterns. The HEK cells also transfected with pmaxGFP plasmid vector to show that the cells on collagen micropatterns could also used in transfection studies.

Conclusion

Taking these together, this novel method is promising for efficient cell culture studies on micropatterened surfaces in the future.

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Physical Explanation of Coupled Cell-Cell Rotational Behavior and Interfacial Morphology: A Particle Dynamics Model

Previous studies have reported persistent rotational behavior between adherent cell-cell pairs cultured on micropatterned substrates, and this rotation is often accompanied by a sigmoidal deflection of the cell-cell interface. Interestingly, the cell-cell rotation runs in the opposite reference frame from what could be expected of single cell locomotion. Specifically, the rotation of the cell pair consists of each individual cell protruding from the inwardly regressive arm of the cell-cell interface, and retracting from the other outwardly protrusive arm. To this author’s knowledge, the cause of this elusive behavior has not yet been clarified. Here, we propose a physical model based on particle dynamics, accounting for actomyosin forcing, viscous dissipation, and cortical tension. The results show that a correlation in actomyosin force vectors leads to both persistent rotational behavior and interfacial deflection in a simulated cell cluster. Significantly, the model, without any artificial cues, spontaneously and consistently reproduces the same rotational reference frame as experimentally observed. Further analyses show that the interfacial deflection depends predominantly on cortical tension, whereas the cluster rotation depends predominantly on actomyosin forcing. Together, these results corroborate the hypothesis that both rotational and morphological phenomena are, in fact, physically coupled by an intracellular torque of a common origin.

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Patterned cell arrays and patterned co-cultures on polydopamine-modified poly(vinyl alcohol) hydrogels

Live cell arrays are an emerging tool that expand traditional 2D in vitro cell culture, increasing experimental precision and throughput. A patterned cell system was developed by combining the cell-repellent properties of polyvinyl alcohol hydrogels with the cell adhesive properties of self-assembled films of dopamine (polydopamine). It was shown that polydopamine could be patterned onto spin-cast polyvinyl alcohol hydrogels by microcontact printing, which in turn effectively patterned the growth of several cell types (HeLa, human embryonic kidney, human umbilical vein endothelial cells (HUVEC) and prostate cancer). The cells could be patterned in geometries down to single-cell confinement, and it was demonstrated that cell patterns could be maintained for at least 3 weeks. Furthermore, polydopamine could be used to modify poly(vinyl alcohol) in situ using a cell-compatible deposition buffer (1 mg mL−1 dopamine in 25 mM tris with a physiological salt balance). The treatment switched the PVA hydrogel from cell repellent to cell adhesive. Finally, by combining microcontact printing and in situ deposition of polydopamine, patterned co-cultures of the same cell type (HeLa/HeLa) and dissimilar cell types (HeLa/HUVEC) were realized through simple chemistry and could be studied over time. The combination of polyvinyl alcohol and polydopamine was shown to be an attractive route to versatile, patterned cell culture experiments with minimal infrastructure requirements and low complexity.

 

 

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Deciphering the Combinatorial Roles of Geometric, Mechanical, and Adhesion Cues in Regulation of Cell Spreading

Deciphering the Combinatorial Roles of Geometric, Mechanical, and Adhesion Cues in Regulation of Cell Spreading | Micropatterns | Scoop.it

Significant effort has gone towards parsing out the effects of surrounding microenvironment on macroscopic behavior of stem cells. Many of the microenvironmental cues, however, are intertwined, and thus, further studies are warranted to identify the intricate interplay among the conflicting downstream signaling pathways that ultimately guide a cell response. In this contribution, by patterning adhesive PEG (polyethylene glycol) hydrogels using Dip Pen Nanolithography (DPN), we demonstrate that substrate elasticity, subcellular elasticity, ligand density, and topography ultimately define mesenchymal stem cells (MSCs) spreading and shape. Physical characteristics are parsed individually with 7 kilopascal (kPa) hydrogel islands leading to smaller, spindle shaped cells and 105 kPa hydrogel islands leading to larger, polygonal cell shapes. In a parallel effort, a finite element model was constructed to characterize and confirm experimental findings and aid as a predictive tool in modeling cell microenvironments. Signaling pathway inhibition studies suggested that RhoA is a key regulator of cell response to the cooperative effect of the tunable substrate variables. These results are significant for the engineering of cell-extra cellular matrix interfaces and ultimately decoupling matrix bound cues presented to cells in a tissue microenvironment for regenerative medicine.

 

 

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Dynamic modulation of small-sized multi-cellular clusters using a cell-friendly photoresist

Dynamic modulation of small-sized multi-cellular clusters using a cell-friendly photoresist | Micropatterns | Scoop.it

Dynamics of small-sized multi-cellular clusters is important for many biological processes including embryonic development and cancer metastasis. Previous methods to fabricate multi-cellular clusters depended on stochastic adhesion and proliferation of cells on defined areas of cell-adhering islands. This made precise control over the number of cells within multi-cellular clusters impossible. Variation in numbers may have minimal effects on the behavior of multi-cellular clusters composed of tens of cells, but would have profound effects on groups with less than ten cells. Herein, we report a new dynamic cell micro-patterning method using a cell-friendly photoresist film by multi-step microscope projection photolithography (MPP). We first fabricated single cell arrays of partially spread cells. Then, by merging neighboring cells, we successfully fabricated multi-cellular clusters with precisely controlled number, composition, and geometry. Using this method, we generated multi-cellular clusters of Madin-Darby canine kidney (MDCK) cells with various numbers and initial geometries; then, systematically investigated the effect of multi-cellular cluster sizes and geometries on their motility behaviors. We found out that the behavior of small-sized multi-cellular clusters was not sensitive to initial configurations, but determined by dynamic force balances among the cells. Initially, the multi-cellular clusters exhibited rounded morphology and minimal translocation probably due to contractility at the periphery of the clusters. For 2-cell and 4-cell clusters, single leaders emerged over time and entire groups aligned and co-migrated as single super-cells. Such coherent behavior did not occur in 8-cell clusters, indicating critical group size led by a single leader may exist. The method developed in the study will be useful for the study of collective migration and multi-cellular dynamics.

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Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains ( = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.

 

 

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Attraction and Repulsion: Cellular Micropatterns on a Graphene Film

Attraction and Repulsion: Cellular Micropatterns on a Graphene Film | Micropatterns | Scoop.it

Graphene has recently attracted a great deal of attention in the scientific community, as evidenced by the Nobel Prize in Physics in 2010. The material is by definition a single-atom thick carbon sheet, which despite its lightness is exceptionally strong. Graphene is also nearly transparent and has excellent electrical conductivity, making it an attractive platform for analyzing cellular activity. However, techniques to chemically modify graphene are limited, which has made it difficult to implement graphene as a viable observational system.

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