Gradient-free directional cell migration in continuous microchannels.

Directing cell movements within 3D channels is a key challenge in biomedical devices and tissue engineering. In two dimensions, closely spaced arrays of asymmetric teardrop islands can intermittently polarize cells and sustain their autonomous directional migration with no gradients. However, in 3D microchannels composed of linearly connected teardrop segments, negligibly low directional bias is observed. Rather than adopt teardrop shapes, cells evade morphological polarization by spreading across multiple teardrop segments, only partly filling each. We demonstrate here that cells can be forced to adopt the shape of individual segments by connecting the segments at an angle to minimize cell spreading across multiple segments. The resulting rhythmic polarization leads to significant directional bias for NIH3T3 fibroblasts, epithelial cells, and even cells whose intracellular signalling have been purposely altered to affect lamellipodia extension (Rac1) and cell polarity (Cdc42). This gradient-free approach to directing cell migration in 3D microchannels may find significant applications in tissue scaffolds and cell on a chip devices.

Motility-based cell sorting by planar cell chromatography.

We report here a new methodology for sorting mammalian cells based on their intrinsic motility on planar substrates, independent of chemoattractants and external fields. This biological analogue of thin layer chromatography consists of arrays of asymmetric adhesive islands on tissue culture dishes that rectify the random movement of cells and direct their migration in a specific direction. We demonstrated the use of planar cell chromatography in the separation of mixtures of 3T3 fibroblasts that express constitutively active Rac1 or RhoA and mixtures of 3T3 fibroblasts and SH-SY5Y neuroblastoma cells.

Differential migration and proliferation of geometrical ensembles of cell clusters.

Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

Steering cell migration using microarray amplification of natural directional persistence.

Cell locomotion plays a key role in embryonic morphogenesis, wound healing, and cancer metastasis. Here we show that intermittent control of cell shape using microarrays can be used to amplify the natural directional persistence of cells and guide their continuous migration along preset paths and directions. The key to this geometry-based, gradient-free approach for directing cell migration is the finding that cell polarization, induced by the asymmetric shape of individual microarray islands, is retained as cells traverse between islands. Altering the intracellular signals involved in lamellipodia extension (Rac1), contractility (RhoA), and cell polarity (Cdc42) alters the speed of fibroblast migration on these micropatterns but does not affect their directional bias significantly. These results provide insights into the role of cell morphology in directional movement and the design of micropatterned materials for steering cellular traffic.

Arresting amphiphilic self-assembly.

Amphiphilic self-assembly has become the basis for a wide gamut of materials and commercial product applications. In many situations however, the best use of self-assembling complex fluids comes when their microstructures can be made permanent. The impetus for a static microstructure can often be such that an alternative non-aqueous media is preferable. Highlighted here is a new approach to capturing self-assembly through replacement of water in traditional complex fluids with sugars to form room temperature complex glasses. Combining solid and liquid properties at the nanoscale, complex glasses have broad potential applications in encapsulation and materials template synthesis.

Alternating polymervesicles.

We demonstrate here that the formation of polymer vesicles is not the exclusive realm of amphiphilic block copolymers. The natural alternating conjugation of hydrophobic alkyl maleates and hydrophilic polyhydroxy vinyl ethers under free-radical polymerization conditions also yields polymers with sufficient backbone amphiphilicity to form vesicles. In contrast to conventional polymersomes, these polymer vesicles have thin flexible shells capable of forming ultra-small unilamellar vesicles in water as confirmed by cryogenic-transmission electron microscopy (cryo-TEM), small-angle neutron scattering (SANS), and dynamic light scattering (DLS). The encapsulation and release characteristics of these alternating polymer vesicles are, however, similar to their surfactant counterparts.

Formation of capillary tube-like structures on micropatterned biomaterials.

The survival of three-dimensional tissue requires a vascular network to provide transport of oxygen and metabolic byproduct. Here, we report a new approach to create capillary blood vessels in vitro on biomaterials suitable for use as scaffolds in engineering tissues. Endothelial cells were cultured on chemical and topographical patterns of micro-sized grooves on gelatin. Selective attachment and spreading of cells within the grooves was ensured by microcontact printing the plateau regions with cell resistant PEG/PLA (polyethyleneglycol-L-polylacticacid). Human microvascular endothelial cells plated on these patterned biomaterials attached and spread exclusively within the grooves. These topographical features promote endothelial cells to form capillary tube-like structures. The results demonstrated that capillary structures formed on biomaterials are useful for engineering vascularized tissues.

Author Correction: Geometric Control of Cell Migration.

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Engineering Dynamic Biointerfaces.

Recent advances in dynamic biointerfaces enable spatiotemporal control over cell position and migration after attachment using substrates that employ chemical, optical, thermal, or electrical triggers. This review focuses on flexible and accessible methods for the fabrication of cellular arrays or co cultures for fundamental studies of cell biology or regenerative medicine.

Alteration of human neuroblastoma cell morphology and neurite extension with micropatterns.

The spatial orientation of nerve cells plays a pivotal role in nerve regeneration. Here we report a new method for regulating neuronal cell morphology and guiding neurite extension on standard tissue culture dishes. Random copolymers of oligoethyleneglycol methacrylate and methacrylic acid [poly(OEGMA-co-MA)], microcontact printed on standard tissue culture dishes, resist cell attachment and remain intact in serum-containing medium for up to 2 weeks. Cell viability assay of SH-SY5Y cells demonstrated that poly(OEGMA-co-MA) on the substrate or in solution has no cytotoxic effect. When retinoic acid was added to SH-SY5Y cells, they extended neurites along the line patterns that are significantly longer than cells cultured on non-patterned culture dishes. The ability to guide neurite extension with micrometer precision is valuable for guiding directional growth of neurites and path finding of regenerating nerves.

Micropatterning proteins and cells on polylactic acid and poly(lactide-co-glycolide).

Techniques for micropatterning proteins and cells on biomaterials are important in tissue engineering applications. Here, we present a method for patterning proteins and cells on poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) substrates that are routinely used as scaffolds in engineering tissues. Poly(oligoethyleneglycol methacrylate) (poly-OEGMA) or poly(oligoethyleneglycol methacrylate-co-methacrylic acid) (poly(OEGMA-co-MA)) was microcontact printed onto substrates to create cell resistant areas. Proteins adsorbed onto the unprinted regions whereas the polymer printed regions effectively repel non-specific protein adsorption. NIH 3T3 fibroblasts remain confined within the patterns on the PLGA and PLA films for up to 2 weeks and aligned their actin cytoskeleton along the line patterns. In comparison to unpatterned cells, fibroblasts confined within line-shaped patterns show fewer actin filaments. This method for controlling the spatial morphology and distribution of cells on synthetic biomaterials could have significant applications in tissue engineering.

Spatially controlled co-culture of neurons and glial cells.

The ability to create and maintain neuron and glial cell co-cultures is important for neuronal regeneration as well as for fundamental studies on neuron and glial cell interactions. We demonstrate here a method for spatially controlling the arrangement of neurons and glial cells. Line patterns of cell resistant, poly(oligoethyleneglycol methacrylate-co-methacrylic acid), was microcontact printed on various substrates to spatially control the attachment of neurons. Neuron-like cells, PC12 and SH-SY5Y cells, were confined within the unprinted line patterns and extended neurites along the line patterns. Subsequent attachment of glial cells was accomplished by converting the originally cell-resistant line patterns of poly(oligoethyleneglycol methacrylate-co-methacrylic acid) to cell adhesive by electrostatic adsorption of cationic poly-lysine, chitosan, or poly(ethyleneimine). This method for creating patterned co-cultures of neuron and glial cells provides a useful tool for investigating neuron-glial cell interactions and has potential applications in the repair or regeneration of nervous systems.

Cell-Substrate Interactions Feedback to Direct Cell Migration along or against Morphological Polarization.

In response to external stimuli, cells polarize morphologically into teardrop shapes prior to moving in the direction of their blunt leading edge through lamellipodia extension and retraction of the rear tip. This textbook description of cell migration implies that the initial polarization sets the direction of cell migration. Using microfabrication techniques to control cell morphologies and the direction of migration without gradients, we demonstrate that after polarization, lamelipodia extension and attachment can feedback to change and even reverse the initial morphological polarization. Cells do indeed migrate faster in the direction of their morphologically polarization. However, feedback from subsequent lamellipodia extension and attachment can be so powerful as to induce cells to reverse and migrate against their initial polarization, albeit at a slower speed. Constitutively active mutants of RhoA show that RhoA stimulates cell motility when cells are guided either along or against their initial polarization. Cdc42 activation and inhibition, which results in loss of directional motility during chemotaxis, only reduces the speed of migration without altering the directionality of migration on the micropatterns. These results reveal significant differences between substrate directed cell migration and that induced by chemotactic gradients.

Cell shape dependent regulation of nuclear morphology.

Recent studies suggest that actin filaments are essential in how a cell controls its nuclear shape. However, little is known about the relative importance of membrane tension in determining nuclear morphology. In this study, we used adhesive micropatterned substrates to alter the cellular geometry (aspect ratio, size, and shape) that allowed direct membrane tension or without membrane lateral contact with the nucleus and investigate nuclear shape remodeling and orientation on a series of rectangular shapes. Here we showed that at low cell aspect ratios the orientation of the nucleus was regulated by actin filaments while cells with high aspect ratios can maintain nuclear shape and orientation even when actin polymerization was blocked. A model adenocarcinoma cell showed similar behavior in the regulation of nuclear shape in response to changes in cell shape but actin filaments were essential in maintaining cell shape. Our results highlight the two distinct mechanisms to regulate nuclear shape through cell shape control and the difference between fibroblasts and a model cancerous cell in cell adhesion and cell shape control.

Directing cell migration in continuous microchannels by topographical amplification of natural directional persistence.

Discrete micropatterns on biomaterial surfaces can be used to guide the direction of mammalian cell movement by orienting cell morphology. However, guiding cell assembly in three-dimensional scaffolds remains a challenge. Here we demonstrate that the random motions of motile cells can be rectified within continuous microchannels without chemotactic gradients or fluid flow. Our results show that uniform width microchannels with an overhanging zigzag design can induce polarization of NIH3T3 fibroblasts and human umbilical vein endothelial cells by expanding the cell front at each turn. These continuous zigzag microchannels can guide the direction of cell movement even for cells with altered intracellular signals that promote random movement. This approach for directing cell migration within microchannels has important potential implications in the design of scaffolds for tissue engineering.

Geometric control of cell migration.

Morphological polarization involving changes in cell shape and redistribution of cellular signaling machinery, initiate the migration of mammalian cells. Golgi complex typically localizes in front of the nucleus, and this frontwards polarization has been proposed to be involved in directional migration. However, the sequence of events remains unresolved. Does Golgi polarization precede directional migration or vice-versa? We address this question by constraining cells to specific areas and shapes then tracking their motile behavior and the spatio-temporal distribution of Golgi apparatus upon release. Results show that while the position of the Golgi complex depends on the cell geometry, the subcellular localization of the Golgi complex does not define the cell's leading edge. Cells constrained within elongated geometries exhibit polarized extension of lamellipodia and upon release, migrate preferentially along the long axis of the cell. Minimally constrained cells released from larger areas however, exhibit retarded migration regardless of lamellipodia protrusion activity.

Rapid Prototyping of Heterotypic Cell-Cell Contacts.

Disparities in cellular behaviour between cultures of a single cell type and heterogeneous co-cultures require constructing spatially-defined arrays of multiple cell types. Such arrays are critical for investigating cellular properties as they exist in vivo. Current methods rely upon covalent surface modification or external physical micromanipulation to control cellular organization on a limited range of substrates. Here, we report a direct approach for creating co-cultures of different cell types by microcontact printing a photosensitive cell resist. The cell-resistant polymer converts to cell adhesive 0 with light exposure, thus the initial copolymer pattern dictates the position of both cell types. This strategy enables straightforward preparation of tailored heterotypic cell-cell contacts on materials ranging from polymers to metallic substrates.

Micropatterning different cell types with microarray amplification of natural directional persistence.

In vivo, different cell types assemble in specific patterns to form functional tissues. Reproducing this process in vitro by designing scaffold materials to direct cells precisely to the right locations at the right time is important for the next generation of biomaterials. Here, using microarray amplification of natural directional persistence (MANDIP), simultaneous assembly of fibroblasts and endothelial cells is demonstrated by directing their long-range migration. Amplification of the directional persistence occurs through morphology-induced polarity and the asymmetric positioning of individual microsized adhesive islands that restrict lamellipodia attachment, and thus migration, to one preset direction. Quantitative analysis of cell migration on different MANDIP designs yields insight to the relative importance of the asymmetric island shapes and their arrangement. The approach enables spatial patterning of different cell types with micrometer-scale precision over large areas for investigation of cell-cell interactions within complex tissue architectures.