Two-step cell patterning on planar and complex curved surfaces by precision spraying of polymers

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.     

The modulation of canine mesenchymal stem cells by nano-topographic cues

Mesenchymal stem cells (MSCs) represent a promising cellular therapeutic for the treatment of a variety of disorders. On transplantation, MSCs interact with diverse extracellular matrices (ECMs) that vary dramatically in topographic feature type, size and surface order. In order to investigate the impact of these topographic cues, surfaces were fabricated with either isotropically ordered holes or anisotropically ordered ridges and grooves. To simulate the biologically relevant nano through micron size scale, a series of topographically patterned substrates possessing features of differing pitch (pitch=feature width+groove width) were created. Results document that the surface order and size of substratum topographic features dramatically modulate fundamental MSC behaviors. Topographically patterned (ridge+groove) surfaces were found to significantly impact MSC alignment, elongation, and aspect ratio. Novel findings also demonstrate that submicron surfaces patterned with holes resulted in increased MSC alignment to adjacent cells as well as increased migration rates. Overall, this study demonstrates that the presentation of substratum topographic cues dramatically influence MSC behaviors in a size and shape dependent manner. The response of MSCs to substratum topographic cues was similar to other cell types that have been studied previously with regards to cell shape on ridge and groove surfaces but differed with respect to proliferation and migration. This is the first study to compare the impact of anisotropically ordered ridge and groove topographic cues to isotropically order holed topographic cues on fundamental MSC behaviors across a range of biologically relevant size scales.     

Photoinitiated alkyne-azide click and radical cross-linking reactions for the patterning of PEG hydrogels

The photolithographical patterning of hydrogels based solely on the surface immobilization and cross-linking of alkyne-functionalized poly(ethylene glycol) (PEG-tetraalkyne) is described. Photogenerated radicals as well as UV absorption by a copper chelating ligand result in the photochemical redox reduction of Cu(II) to Cu(I). This catalyzes the alkyne-azide click reaction to graft the hydrogels onto an azide-functionalized plasma polymer (N(3)PP) film. The photogenerated radicals were also able to abstract hydrogen atoms from PEG-tetraalkyne to form poly(α-alkoxy) radicals. These radicals can initiate cross-linking by addition to the alkynes and intermolecular recombination to form the PEG hydrogels. Spatially controlling the two photoinitiated reactions by UV exposure through a photomask leads to surface patterned hydrogels, with thicknesses that were tunable from tens to several hundreds of nanometers. The patterned PEG hydrogels (ca. 60 μm wide lines) were capable of resisting the attachment of L929 mouse fibroblast cells, resulting in surfaces with spatially controlled cell attachment. The patterned hydrogel surface also demonstrated spatially resolved chemical functionality, as postsynthetic modification of the hydrogels was successfully carried out with azide-functionalized fluorescent dyes via subsequent alkyne-azide click reactions.     

Cell patterning using magnetite nanoparticles and magnetic force

Technologies for fabricating functional tissue architectures by patterning cells precisely are highly desirable for tissue engineering. Although several cell patterning methods such as microcontact printing and lithography have been developed, these methods require specialized surfaces to be used as substrates, the fabrication of which is time consuming. In the present study, we demonstrated a simple and rapid cell patterning technique, using magnetite nanoparticles and magnetic force, which enables us to allocate cells on arbitrary surfaces. Magnetite cationic liposomes (MCLs) developed in our previous study were used to magnetically label the target cells. When steel plates placed on a magnet were positioned under a cell culture surface, the magnetically labeled cells lined on the surface where the steel plate was positioned. Patterned lines of single cells were achieved by adjusting the number of cells seeded, and complex cell patterns (curved, parallel, or crossing patterns) were successfully fabricated. Since cell patterning using magnetic force may not limit the property of culture surfaces, human umbilical vein endothelial cells (HUVECs) were patterned on Matrigel, thereby forming patterned capillaries. These results suggest that the novel cell patterning methodology, which uses MCLs, is a promising approach for tissue engineering and studying cell-cell interactions in vitro.     

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. Microcontact printing (μCP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.^1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 μm.^10-16In contrast to traditional printing, inkless μCP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (<200 nm) features.^17-23 However, up till now, inkless μCP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many μCP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.     

Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor

A strategy for patterned cell adhesion based on chemical surface modification is presented. To confine cell adhesion to specific locations, an engineered surface for high-contrast protein adsorption and, hence, cell attachment has been developed. Surface functionalization is based on selective molecular-assembly patterning (SMAP). An amine-terminated self-assembled monolayer is used to define areas of cell adhesion. A protein-repellent grafted copolymer, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), is used to render the surrounding silicon dioxide resistant to protein adsorption. X-ray photoelectron spectroscopy, scanning ellipsometry and fluorescence microscopy techniques were used to monitor the individual steps of the patterning process. Successful guided growth using these layers is demonstrated with primary neonatal rat cardiomyocytes, up to 4 days in vitro, and with the HL-1 cardiomyocyte cell line, up to 7 days in vitro. The advantage of the presented method is that high-resolution engineered surfaces can be realized using a simple, cost-effective, dip-and-rinse process. The technique has been developed for application on a CMOS cell-based biosensor, which comprises an array of microelectrodes to extracellularly record electrical activity from cardiomyocytes.     

Topographical and physicochemical modification of material surface to enable patterning of living cells.

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Tissue scaffold surface patterning for clinical applications

Patterned scaffold surfaces provide a platform for highly defined cellular interactions, and have recently taken precedence in tissue engineering. Despite advances in patterning techniques and improved tissue growth, no clinical studies have been conducted for implantation of patterned biomaterials. Four major clinical application fields where patterned materials hold great promise are antimicrobial surfaces, cardiac constructs, neurite outgrowth, and stem cell differentiation. Specific examples include applications of patterned materials to (i) counter infection by antibiotic resistant bacteria, (ii) establish proper alignment and contractile force of regrown cardiac cells for repairing tissue damaged by cardiac infarction, (iii) increase neurite outgrowth for central nervous system wound repair, and (iv) host differentiated stem cells while preventing reversion to a pluripotent state. Moreover, patterned materials offer unique advantages for artificial implants which other constructs cannot. For example, by inducing selective cell adhesion using topographical cues, patterned surfaces present cellular orientation signals that lead to functional tissue architectures. Mechanical stimuli such as modulus, tension, and material roughness are known to influence tissue growth, as are chemical stimuli for cell adhesion. Scaffold surface patterns allow for control of these mechanical and chemical factors. This review identifies research advances in scaffold surface patterning, in light of pressing clinical needs requiring organization of cellular interactions.     

Topographical and Physicochemical Modification of Material Surface to Enable Patterning of Living Cells

ABSTRACT:?

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Preparation and applications of biofunctionalized,supported lipid bilayers and vesicles

Biofunctional surfaces require advanced design and preparation to match the (bio)recognition ability of biological systems [1]. This requires combined topographic, chemical and visco-elastic surface patterns to match proteins at the nm scale and cells at the micrometer scale. One example of biochemical functionalization, presented here, and which is of both fundamental and application interest, is supported biomimectic (cell)membranes. Specifically we describe preparation and applications of supported phospholipid membranes, which can be made on certain surfaces from unilamellar, 25–200 nm vesicles. On SiO2 at normal pH and with neutral lipids, the vesicles first adsorb intact, and then undergo a phase transformation to a supported bilayer. We have studied the coverage-, vesicle size-, and T-dependence of this process [2], using QCM-D [3], AFM, and SPR. When SiO2 is replaced by TiO2, vesicles adsorb intact. A surface pre-covered with intact vesicles, can be AFM patterned into areas with bilayer, vesicles, and empty surface patches [4]. The results depend critically on AFM tip interaction with vesicle and bilayer, which has been modeled by Monte Carlo simulations [5]. These biomembranes are inert towards protein adsorption [6] and cell attachement [7], which opens up for various applications. Addition of functional molecules, allows sensor functions [8]. Another application is functionalized membranes for surface-specific (stem) cell interactions [9].     

Controlled Cell Patterning on Bioactive Surfaces with Special Wettability

The ability to control cell patterning on artificial substrates with various physicochemical properties is of essence for important implications in cytology and biomedical fields.Despite extensive progress,the ability to control the cell-surface interaction is complicated by the complexity in the physiochemical features ofbioactive surfaces.In particular,the manifestation of special wettability rendered by the combination of surface roughness and surface chemistry further enriches the cell-surface interaction.Herein we investigated the cell adhesion behaviors of Circulating Tumor Cells (CTCs) on topographically patterned but chemically homogeneous surfaces.Hamessing the distinctive cell adhesion on surfaces with different topography,we further explored the feasibility of controlled cell patterning using periodic lattices of alternative topographies.We envision that our method provides a designer's toolbox to manage the extracellular environment.     

Comparative Endothelial Cell Response on Topographically Patterned Titanium and Silicon Substrates with Micrometer to Sub-Micrometer Feature Sizes

In this work, we evaluate the in vitro response of endothelial cells (EC) to variation in precisely-defined, micrometer to sub-micrometer scale topography on two different substrate materials, titanium (Ti) and silicon (Si). Both substrates possess identically-patterned surfaces composed of microfabricated, groove-based gratings with groove widths ranging from 0.5 to 50 µm, grating pitch twice the groove width, and groove depth of 1.3 µm. These specific materials are chosen due to their relevance for implantable microdevice applications, while grating-based patterns are chosen for the potential they afford for inducing elongated and aligned cellular morphologies reminiscent of the native endothelium. Using EA926 cells, a human EC variant, we show significant improvement in cellular adhesion, proliferation, morphology, and function with decreasing feature size on patterned Ti substrates. Moreover, we show similar trending on patterned Si substrates, albeit to a lesser extent than on comparably patterned Ti substrates. Collectively, these results suggest promise for sub-micrometer topographic patterning in general, and sub-micrometer patterning of Ti specifically, as a means for enhancing endothelialization and neovascularisation for novel implantable microdevice applications.     

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.     

Photolithographic patterning of C2C12 myotubes using vitronectin as growth substrate in serum-free medium

The C2C12 cell line is frequently used as a model of skeletal muscle differentiation. In our serum-free defined culture system, differentiation of C2C12 cells into myotubes required surface-bound signals such as substrate-adsorbed vitronectin or laminin. On the basis of this substrate requirement of myotube formation, we developed a photolithography-based method to pattern C2C12 myotubes, where myotubes formed exclusively on vitronectin surface patterns. We have determined that the optimal line width to form single myotubes is approximately 30 mum. To illustrate a possible application of this method, we patterned myotubes on the top of commercial substrate-embedded microelectrodes. In contrast to previous experiments where cell patterning was achieved by selective attachment of the cells to patterned surfaces in a medium that contained all of the factors necessary for differentiation, this study illustrates that surface patterning of a signaling molecule, which is essential for skeletal muscle differentiation in a defined system, can result in the formation of aligned myotubes on the patterns. This technique is being developed for applications in cell biology, tissue engineering, and robotics.     

Micropatterning of biomolecules on glass surfaces modified with various functional groups using photoactivatable biotin

Biomolecule patterning by photolithographic methods has considerable advantages because a large number of different biomolecules can be assembled on a spatial area by a combinatorial method and complex biomolecule patterning can be created in situ in closed environments such as microfluidic channels. Here, a photobiotin was used as the photoactivatable reagent to create patterned arrays of biomolecules. The variability of photobiotin deposition on glass substrates modified with a variety of materials having carboxyl, lysine, aldehyde, amine groups, and BSA (bovine serum albumin) was characterized by subsequent derivatization with Cy3-labeled streptavidin. The fluorescence images of the photobiotin patterned glass surfaces showed that the BSA/aldehyde-coated glass could be considered as the most appropriate substrate to immobilize photobiotin, in view of the homogeneous immobilization of biomolecules with high density in defined regions and the reduction of nonspecific binding to the surface. In streptavidin equilibrium adsorption assays, the maximum amount of streptavidin-Cy3 bound to the BSA/aldehyde-coated glass surface continued to rise with increasing streptavidin-Cy3 concentration until 12.0 microg/mL was reached and the surface then became saturated. Also, a line array of biotin-labeled single-strand probe DNAs was created on the BSA/aldehyde-coated glass by photolysis of photobiotin through a slit-type mask and biotin/streptavidin/biotin chemistry, extended to a quantitative measurement of the concentrations of target DNA. The results of target DNA analysis showed linearity over a wide range from 0.5 ng/mL to 5 microg/mL and were reproducible.     

Spatial distribution of mammalian cells dictated by material surface chemistry

Anisotropic cell culture surfaces patterned with amino and alkylsilanes can guide cell distribution and provide an approach to study important processes involved in tissue engineering, such as cell attachment and locomotion. By combining photolithographic and silane coupling techniques, glass coverslips were patterned with either n-octadecyldimethylchlorosilane (ODDMS) or dimethyldichlorosilane (DMS), and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS). The alkylsilanes, theoretically, have similar methyl and methylene groups exposed at the surface but different structures, with DMS being amorphous and ODDMS ordered. Neuroblastoma cells, osteosarcoma cells, and fibroblasts plated on surfaces patterned with EDS/ODDMS and EDS/DMS specifically localized on the EDS regions, but distributed randomly on ODDMS/DMS patterned surfaces. The preferential assembly of cells onto EDS regions did not depend on the structure of the adjacent alkylsilane regions and was a time-dependent process. Angle dependent x-ray photoelectron spectroscopy (XPS) and contact angle measurements indicated that EDS was immobilized on glass as a fractional hydrophilic monolayer, and ODDMS and DMS were bound as patchy amorphous hydrophobic multilayers. Neither surface coverage nor thickness of the overlayer seemed to be as important as surface chemistry, or charge, in guiding mammalian cell distribution. These results are consistent with the concept that mammalian cells attach to and are guided by positively charged surfaces.     

Engineered antifouling microtopographies: mapping preferential and inhibitory microenvironments for zoospore attachment

An algorithm was developed and implemented to map the locations of attached spores of Ulva linza on patterned surfaces. Using this mapping algorithm, spore preference among regions within a pattern can be detected and quantified. Settlement maps of spores on patterned topographies from several assays showed clear preferences in spore settlement. Over 94% of the spores attached within the depressed regions on the surfaces, including a surface containing pits instead of protruding features. The spores attached primarily at the intersections of several features, with over half and up to 96% of spores settling in these regions. The highest spore densities occurred at intersections where the features were most dissimilar. In contrast, the location of attached beads on the surfaces was nearly uniform across the surface. Identification of preferential attachment locations allows for the study of localized properties that influence cell behavior and aids in the development of new surfaces to control cell–surface interactions.     

Clusters of ligands on dendrimer surfaces

The development of methodology that is designed to allow a significant increase in the patterning and in the functionalization of the dendrimer is the ultimate goal of the research described here. Glycoside clusters based on TRIS were formed using click chemistry and were attached to PAMAM dendrimers. A series of dendrimers bearing tris-mannoside and an ethoxyethanol group was synthesized, and the binding interactions of these dendrimers with Concanavalin A were evaluated using inhibition ELISAs. The results of the inhibition ELISAs suggest that tris-mannoside clusters can replace individual sugars on the dendrimer without loss of function. Since tris-mannoside clustering allows for a redistribution of the dendrimers’ surface functionalities, from this chemistry one can envision patterned dendrimers that incorporate multiple groups to increase the function and utility of the dendrimer.     

Nano rough micron patterned titanium for directing osteoblast morphology and adhesion

Previous studies have demonstrated greater functions ofosteoblasts (bone-forming cells) on nanophase compared with conventional metals. Nanophase metals possess a biologically inspired nanostructured surface that mimics the dimensions of constituent components in bone, including collagen and hydroxyapatite. Not only do these components possess dimensions on the nanoscale, they are aligned in a parallel manner creating a defined orientation in bone. To date, research has yet to evaluate the effect that organized nanosurface features can have on the interaction of osteoblasts with material surfaces. Therefore, to determine if surface orientation of features can mediate osteoblast adhesion and morphology, this study investigated osteoblast function on patterned titanium substrates containing alternating regions of micron rough and nano rough surfaces prepared by novel electron beam evaporation techniques. This study was also interested in determining whether or not the size of the patterned regions had an effect on osteoblast behavior and alignment. Results indicated early controlled osteoblast alignment on these patterned materials as well as greater osteoblast adhesion on the nano rough regions of these patterned substrates. Interestingly, decreasing the width of the nano rough regions (from 80 microm to 22 microm) on these patterned substrates resulted in a decreased number of osteoblasts adhering to these areas. Changes in the width of the nano rough regions also resulted in changes in osteoblast morphology, thus, suggesting there is an optimal pattern dimension that osteoblasts prefer. In summary, results of this study provided evidence that aligned nanophase metal features on the surface of titanium improved early osteoblast functions (morphology and adhesion) promising for their long term functions, criteria necessary to improve orthopedic implant efficacy.     

Rectified Cell Migration on Saw-Like Micro-Elastically Patterned Hydrogels with Asymmetric Gradient Ratchet Teeth

To control cell motility is one of the essential technologies for biomedical engineering. To establish a methodology of the surface design of elastic substrate to control the long-range cell movements, here we report a sophisticated cell culture hydrogel with a micro-elastically patterned surface that allows long-range durotaxis. This hydrogel has a saw-like pattern with asymmetric gradient ratchet teeth, and rectifies random cell movements. Durotaxis only occurs at boundaries in which the gradient strength of elasticity is above a threshold level. Consequently, in gels with unit teeth patterns, durotaxis should only occur at the sides of the teeth in which the gradient strength of elasticity is above this threshold level. Therefore, such gels are expected to support the long-range biased movement of cells via a mechanism similar to the Feynman-Smoluchowski ratchet, i.e., rectified cell migration. The present study verifies this working hypothesis by using photolithographic microelasticity patterning of photocurable gelatin gels. Gels in which each teeth unit was 100–120 µm wide with a ratio of ascending:descending elasticity gradient of 1:2 and a peak elasticity of ca. 100 kPa supported the efficient rectified migration of 3T3 fibroblast cells. In addition, long-range cell migration was most efficient when soft lanes were introduced perpendicular to the saw-like patterns. This study demonstrates that asymmetric elasticity gradient patterning of cell culture gels is a versatile means of manipulating cell motility.