Tuning cell adhesion on gradient poly(2-hydroxyethyl methacrylate)-grafted surfaces

A simple yet versatile method was developed to prepare a low-density polymerization initiator gradient, which was combined with surface-initiated atom transfer radical polymerization (ATRP) to produce a well-defined poly(2-hydroxyethyl methacrylate) (HEMA) gradient substrate. A smooth variation in film thickness was measured across the gradient, ranging from 20 A to over 80 A, but we observed a nonmonotonic variation in water contact angle. Fits of X-ray reflectivity profiles suggested that at the low graft density end, the polymer chain structure was in a "mushroom" regime, while the polymer chains at high graft density were in a "brush" regime. It was found that the "mushroom" region of the gradient could be made adhesive to cells by adsorbing adhesion proteins, and cell adhesion could be tuned by controlling the density of the polymer grafts. Fibroblasts were seeded on gradients precoated with fibronectin to test cellular responses to this novel substrate, but it was found that cell adhesion did not follow the expected trend; instead, saturated cell adhesion and spreading was found at the low grafting density region.     

Photoactivation of a substrate for cell adhesion under standard fluorescence microscopes

Cell-culturing substrates where cell adhesion can be switched on by external stimuli during cell cultivation are useful scaffolds for tissue engineering, cell-based drug screening, and fundamental cellular studies. Here, we show a new strategy for photoactivation of a substrate for cell adhesion under standard fluorescence microscopes. A glass substrate chemically modified with an alkylsiloxane having a photocleavable 2-nitrobenzyl group was coated with bovine serum albumin to prevent cell adhesion. Upon irradiation under a fluorescence microscope, the protein was replaced with fibronectin, which made the irradiated region cell-adhesive. Subsequent seeding of HEK293 or COS7 cells produced patterns corresponding to the irradiated patterns. We succeeded for the first time in positioning single cells in proximity to cultivating single cells. The present method provides a general strategy for positioning single cells of same or different types at any locations on the substrate and will be useful for studying cell-cell interactions.     

Generation of oxygen gradients in microfluidic devices for cell culture using spatially confined chemical reactions

This paper reports a microfluidic device capable of generating oxygen gradients for cell culture using spatially confined chemical reactions with minimal chemical consumption. The microfluidic cell culture device is constructed by single-layer polydimethylsiloxane (PDMS) microfluidic channels, in which the cells can be easily observed by microscopes. The device can control the oxygen gradients without the utilization of bulky pressurized gas cylinders, direct addition of oxygen scavenging agents, or tedious gas interconnections and sophisticated flow control. In addition, due to the efficient transportation of oxygen within the device using the spatially confined chemical reactions, the microfluidic cell culture device can be directly used in conventional cell incubators without altering their gaseous compositions. The oxygen gradients generated in the device are numerically simulated and experimentally characterized using an oxygen-sensitive fluorescence dye. In this paper, carcinomic human alveolar basal epithelial (A549) cells have been cultured in the microfluidic device with a growth medium and an anti-cancer drug (Tirapazamine, TPZ) under various oxygen gradients. The cell experiment results successfully demonstrate the hyperoxia-induced cell death and hypoxia-induced cytotoxicity of TPZ. In addition, the results confirm the great cell compatibility and stable oxygen gradient generation of the developed device. Consequently, the microfluidic cell culture device developed in this paper is promising to be exploited in biological labs with minimal instrumentation to study cellular responses under various oxygen gradients.     

Photocontrol of cell adhesion on amino-bearing surfaces by reversible conjugation of poly(ethylene glycol) via a photocleavable linker

Dynamic control of cell adhesion on substrates is a useful technology in tissue engineering and basic biology. This paper describes a method for the control of cell adhesion on amino-bearing surfaces by reversible conjugation of an anti-fouling polymer, poly(ethylene glycol) (PEG), via a newly developed photocleavable linker, 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate (1). This molecule has alkyne and succinimidyl carbonate at each end, which are connected by photocleavable 2-nitrobenzyl ester. Under this molecular design, the molecule crosslinked azides and amines, whose linkage cleaved upon application of near-UV light. By using aminosilanised glass and silicon as model substrates, we studied their reversible surface modification with PEG-azide (M(w) = 5000) based on contact angle measurements, ellipsometry, and AFM morphological observations. Protein adsorption and cell adhesion dramatically changed by PEGylation and the following irradiation, which can be used for cellular patterning. Also, the capability of the substrate to change cell adhesiveness by photoirradiation during cell cultivation was demonstrated by inducing cell migration. We believe this method will be useful for dynamic patterning of cells on protein-based scaffolds.     

Patterning the surface cytophobicity of an albumin-physisorbed substrate by electrochemical means

Spatiotemporal control of surface properties under physiological conditions such as those found in culture media is an important technique in fundamental cell biology, tissue engineering, and cell-based bioelectronics. To this end, we have developed a mild, wet cellular micropatterning technique. The principle of the technique is based on the fact that the cell-repellant property of the albumin-coated substrate rapidly switches to cell-adhesive upon exposure to the reactive oxidizing agent, electrochemically generated hypobromous acid. Herein, we report the effect of the hypobromous acid on serum albumin physisorbed on a hydrophobic substrate. It was found that albumin molecules detach from the substrate by application of the oxidizing agent, resulting in exposure of the underlying hydrophobic surface to the liquid phase. The adsorption of extracellular matrix proteins such as fibronectin onto the hydrophobic surface induces cell adhesion and growth.     

Polyelectrolyte multilayers with a tunable Young's modulus: influence of film stiffness on cell adhesion

Mechanical properties of model and natural gels have recently been demonstrated to play an important role in various cellular processes such as adhesion, proliferation, and differentiation, besides events triggered by chemical ligands. Understanding the biomaterial/cell interface is particularly important in many tissue engineering applications and in implant surgery. One of the final goals would be to control cellular processes precisely at the biomaterial surface and to guide tissue regeneration. In this work, we investigate the substrate mechanical effect on cell adhesion for thin polyelectrolyte multilayer (PEM) films, which can be easily deposited on any type of material. The films were cross linked by means of a water-soluble carbodiimide (EDC), and the film elastic modulus was determined using the AFM nanoindentation technique with a colloidal probe. The Young's modulus could be varied over 2 orders of magnitude (from 3 to 400 kPa) for wet poly(L-lysine)/hyaluronan (PLL/HA) films by changing the EDC concentration. The chemical changes upon cross linking were characterized by means of Fourier transform infrared spectroscopy (FTIR). We demonstrated that the adhesion and spreading of human chondrosarcoma cells directly depend on the Young's modulus. These data indicate that, besides the chemical properties of the polyelectrolytes, the substrate mechanics of PEM films is an important parameter influencing cell adhesion and that PEM offer a new way to prepare thin films of tunable mechanical properties with large potential biomedical applications including drug release.     

Generation of static and dynamic patterned co-cultures using microfabricated parylene-C stencils

Many biological processes, such as stem cell differentiation, wound healing and development, involve dynamic interactions between cells and their microenvironment. The ability to control these dynamic processes in vitro would be potentially useful to fabricate tissue engineering constructs, study biological processes, and direct stem cell differentiation. In this paper, we used a parylene-C microstencil to develop two methods of creating patterned co-cultures using either static or dynamic conditions. In the static case, embryonic stem (ES) cells were co-cultured with fibroblasts or hepatocytes by using the reversible sealing of the stencil on the substrate. In the dynamic case, ES cells were co-cultured with NIH-3T3 fibroblasts and AML12 hepatocytes sequentially by engineering the surface properties of the stencil. In this approach, the top surface of the parylene-C stencil was initially treated with hyaluronic acid (HA) to reduce non-specific cell adhesion. The stencil was then sealed on a substrate and seeded with ES cells which adhered to the underlying substrate through the holes in the membrane. To switch the surface properties of the parylene-C stencils to cell adhesive, collagen was deposited on the parylene-C surfaces. Subsequently, a second cell type was seeded on the parylene-C stencils to form a patterned co-culture. This group of cells was removed by peeling off the parylene-C stencils, which enabled the patterning of a third cell type. Although the static patterned co-culture approach has been demonstrated previously with a variety of methods, layer-by-layer modification of microfabricated parylene-C stencils enables dynamic patterning of multiple cell types in sequence. Thus, this method is a promising approach to engineering the complexity of cell-cell interactions in tissue culture in a spatially and temporally regulated manner.     

Spatiotemporal control of cell adhesion on a self-assembled monolayer having a photocleavable protecting group

Control of cell adhesion is a key technology for cell-based drug screening and for analyses of cellular processes. We developed a method to spatiotemporally control cell adhesion using a photochemical reaction. We prepared a cell-culturing substrate by modifying the surface of a glass coverslip with a self-assembled monolayer of an alkylsiloxane having a photocleavable 2-nitrobenzyl group. Bovine serum albumin (BSA) was adsorbed onto the substrate to make the surface inert to cell adhesion. When exposed to UV light, the alkylsiloxane underwent a photocleavage reaction, leading to the release of BSA from the surface. Fibronectin, a protein promoting cell adhesion, was added to cover the irradiated regions and made them cell-adhesive. Seeding of cells on this substrate resulted in their selective adhesion to the illuminated regions. By controlling the sizes of the illuminated regions, we formed cell-adhesive spots smaller than single cells and located focal adhesions of the cells. Moreover, by subsequently illuminating the region alongside the cells patterned on the substrate in advance, we released their geometrical confinements and induced migration and proliferation. These manipulations were conducted under a conventional fluorescence microscope without any additional instruments. The present method of cell manipulation will be useful for cell biological studies as well as for the formation of cell arrays.     

A biological breadboard platform for cell adhesion and detachment studies

The dynamic nature of cell adhesion and detachment, which plays a critical role in a variety of physiological and pathological phenomena, still remains unclear. This motivates the pursuit of controllable manipulation of cell adhesion and detachment for a better understanding of cellular dynamics. Here we present an addressable, multifunctional, and reusable platform, termed the biological breadboard (BBB), for spatiotemporal manipulation of cell adhesion and detachment at cellular and subcellular levels. The BBB, composed of multiple gold electrodes patterned on a Pyrex substrate, is surface-modified with arginine-glycine-aspartic acid terminated thiol (RTT) and polyethylene glycol (PEG) to achieve a cell-adhesive surface on the gold electrodes and a cell-resistive surface on the Pyrex substrate, respectively. Cell adhesion is regulated by the steric repulsion of PEG chains, while cell detachment is controlled by the reductive desorption of a gold-thiol self-assembled monolayer (SAM) at an activation potential of -0.90 to -1.65 V. Experimental characterizations using NIH 3T3 fibroblasts are presented to demonstrate the utility of our device.     

Evaluation of substrata effect on cell adhesion properties using freestanding poly(L-lactic acid) nanosheets

Investigation of the interactions between cells and material surfaces is important not only for the understanding of cell biology but also for the development of smart biomaterials. In this study, we investigated the substrate-related effects on the interaction between cell and polymeric ultrathin film (nanosheet) by modulating the mechanical properties of the nanosheet with a metal substrate or mesh. A freestanding polymeric nanosheet with tens-of-nanometers thickness composed of poly(L-lactic acid) (PLLA nanosheet) was fabricated by combination of a spin-coating technique and a water-soluble sacrificial layer. The freestanding PLLA nanosheet was collected on a stainless steel mesh (PLLA-mesh) and subsequently used for cell adhesion studies, comparing the results to the ones on a control SiO(2) substrate coated with an ultrathin layer of PLLA (PLLA-substrate). The adhesion of rat cardiomyocytes (H9c2) was evaluated on both samples after 24 h of culture. The PLLA-mesh with the tens-of-nanometers thick nanosheets induced an anisotropic adhesion of H9c2, while H9c2 on the PLLA-substrate showed an isotropic adhesion independent from the nanosheet thickness. Interestingly, an increment in the nanosheet thickness in the PLLA-mesh samples reduced the cellular anisotropy and led to a similar morphology to the PLLA-substrate. Considering the huge discrepancy of Young's modulus between PLLA nanosheet (3.5-4.2 GPa) and metal substrate (hundreds of GPa), cell adhesion was mechanically regulated by the Young's modulus of the underlying substrate when the thickness of the PLLA nanosheet was tens of nanometers. Modulation of the stiffness of the polymeric nanosheet by utilizing a rigid underlying material will allow the constitution of a unique cell culture environment.     

Measurement of cell migration on surface-bound fibronectin gradients

A novel technique for the quantitative observation of cell migration along linear gradient substrates functionalized with adhesive proteins is presented. Gradients of the cell adhesion molecule fibronectin are generated by the cross diffusion of functionalizable alkanethiols on gold and characterized by X-ray photoelectron spectroscopy and surface plasmon resonance. Two distinct migration assays are described that characterize the movement of either sparsely populated noncontacting cells or a confluent monolayer of cells into free space. The drift speed of bovine aortic endothelial cells is measured and shown to increase along a fibronectin gradient when compared to a uniform control substrate using both assays. The results of these experiments establish reproducible conditions for studies of cell migration on gradients of surface-bound ligands.     

Multifunctional mixed SAMs that promote both cell adhesion and noncovalent DNA immobilization

The ability of DNA strands to influence cellular gene expression directly and to bind with high affinity and specificity to other biological molecules (e.g., proteins and target DNA strands) makes them a potentially attractive component of cell culture substrates. On the basis of the potential importance of immobilized DNA in cell culture and the well-defined characteristics of alkanethiol self-assembled monolayers (SAMs), the current study was designed to create multifunctional SAMs upon which cell adhesion and DNA immobilization can be independently modulated. The approach immobilizes the fibronectin-derived cell adhesion ligand Arg-Gly-Asp-Ser-Pro (RGDSP) using carbodiimide activation chemistry and immobilizes DNA strands on the same surface via cDNA-DNA interactions. The surface density of hexanethiol-terminated DNA strands on alkanethiol monolayers (30.2-69.2 pmol/cm2) was controlled using a backfill method, and specific target DNA binding on cDNA-containing SAMs was regulated by varying the soluble target DNA concentration and buffer characteristics. The fibronectin-derived cell adhesion ligand GGRGDSP was covalently linked to carboxylate groups on DNA-containing SAM substrates, and peptide density was proportional to the amount of carboxylate present during SAM preparation. C166-GFP endothelial cells attached and spread on mixed SAM substrates and cell adhesion and spreading were specifically mediated by the immobilized GGRGDSP peptide. The ability to control the characteristics of noncovalent DNA immobilization and cell adhesion on a cell culture substrate suggests that these mixed SAMs could be a useful platform for studying the interaction between cells and DNA.     

Patterning chemical stimulation of reconstructed neuronal networks

A spatially resolved delivery of substances integrated with cell culture substrates shows promise for application in pharmacological assays, bioanalytical studies on cell signaling pathways and cell-based biosensors, where control over the extracellular biochemical environment with a cellular resolution is desirable. In this work, we studied a biohybrid system where rat embryonic cortical neuronal networks are reconstructed on microstructured silicon chips and interfaced to microfluidics. The design of cell-cell and cell-medium interactions in confined geometries is presented. We developed an aligned microcontact printing technique (AμCP) for poly(lysine)-extracellular matrix proteins on microstructured chips, which allows a high degree of geometrical control over the network architecture and alignment of the neuronal network with the microfluidic features of a substrate. Spatially resolved on-chip delivery of compounds with a cellular resolution is demonstrated by chemical stimulation of patterned rat cortical neurons within a network with a number of solutions of excitatory neurotransmitter glutamate delivered via microfluidics. The combination of the system described with a patch-clamp technique allowed both modulation of the biochemical environment on a cellular level and the monitoring of electrophysiological properties in the reconstructed rat embryonic cortical networks changed by this microenvironment.     

Orthogonal surface-grafted polymer gradients: A versatile combinatorial platform

Orthogonal polymer brush gradients are assemblies of surface-anchored macromolecules, in which two material properties of the grafted chains (e.g., grafting density, molecular weight) vary independently in orthogonal directions. Here, we describe the formation and applications of two such orthogonal assemblies, involving: (1) molecular weight and grafting density (MW/σ) gradients of a given polymer and (2) molecular weight gradients (MW1/MW2), of two different polymers. Each point on orthogonal gradient substrate represents a unique combination of the two surface properties being varied, thus facilitating systematic investigation of a phenomenon that depends on the two said properties. We illustrate this point by employing orthogonal structures to study systematically: (1) formation of polymer brush-nanoparticle composite assemblies, (2) protein adsorption and cell adhesion, and (3) chain conformations in tethered diblock copolymers exposed to selective solvents. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 3384–3394, 2005     

Short peptides enhance single cell adhesion and viability on microarrays

Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability, and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and, thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, whereas the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells.     

Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus

Cell adhesion and orientation are important for both natural and engineered tissues to fully achieve physiologic functions. Based on diverse cellular responses induced by electrical stimulus on 2D substrate, we applied non-invasive electrical stimulus to regulate cell adhesion and orientation of bone marrow-derived mesenchymal stem cells (MSCs) and fibroblasts in a reconstituted 3D collagen-based scaffold. While fibroblasts were induced to reorient perpendicularly in response to direct current electrical stimulus, rat MSCs showed only slight changes in cell reorientation. Multiphoton microscopy revealed that rat MSCs exhibited much stronger 3D adhesion, which appears to resist cell reorientation. Only in response to a large electrical stimulus (e.g., 10 V/cm), collagen fibers around rat MSCs became disconnected and loosely reorganized. In contrast, the collagen fibers surrounding the fibroblasts were entangled in a random network and became preferentially aligned in the direction of the electrical stimulus. When incubated with integrin antibodies, both fibroblasts and rat MSCs failed to respond to electrical stimulus, providing evidence that integrin-dependent molecular mechanisms are involved in 3D cell adhesion and orientation. Elucidation of physical regulation of 3D cell adhesion and orientation may offer a novel approach in controlling cell growth and differentiation and could be useful for stem cell-based therapeutic application and engineering tissue constructs.     

Characterizing capsule-substrate adhesion in presence of osmosis

The adhesion of a cellular entity (liquid-filled microcapsule) to a flat glass substrate in response to an osmosis change is studied. A sensitive microscope visualization instrument has been developed to measure the cell–substrate contact area and inflated capsule volume. A theoretical model is developed to quantitatively correlate the adhesion energy to the contact area and osmotic inflation of cell volume. The results show that the contact area increased with increasing adhesion energy, while it shrank in dimension as cell inflation was enlarged. This observed phenomenon is consistent with the theoretical prediction. This work demonstrates the possibility of obtaining quantitative interfacial adhesion energy by using the present technique and represents the first step in extending this approach to study more complicated system such as cell–substrate interaction.     

Quantitatively controlled in situ formation of hydrogel membranes in microchannels for generation of stable chemical gradients

The in situ formation of membranes in microfluidic channels has been given attention because of their great potential in the separation of components, cell culture support for tissue engineering, and molecular transport for generation of chemical gradients. Among these, the porous membranes in microchannels are vigorously applied to generate stable chemical gradients for chemotaxis-dependent cell migration assays. Previous work on the in situ fabrication of membranes for generating the chemical gradient, however, has had several disadvantages, such as fluid leaking, uncontrollable membrane thickness, need of extra equipment, and difficulty in realizing stable interfacial layers. In this paper, we report a novel technique for the in situ formation of membranes within microchannels using enzymatically crosslinkable hydrogels and microfluidic techniques. The thickness of the membrane can be controlled quantitatively by adjusting the crosslinking reaction time and velocity of the microfluidics. By using these techniques, parallel dual hydrogel membranes were prepared within microchannels and these were used for the generation of stable concentration gradients. Moreover, the migration of Salmonella typhimurium was monitored to validate the efficacy of the chemical gradients. These results suggest that our in situ membrane system can be used as a simple platform to understand many cellular activities, including cell adhesion and migration directed by chemotaxis or complex diffusions from biological fluids in three-dimensional microstructures.     

Microelectrochemical approach to induce local cell adhesion and growth on substrates

The nature of an albumin-coated substrate that blocks protein adsorption and cell adhesion was rapidly switched to cell-adhesive by exposure to an oxidizing agent such as HBrO. This finding has enabled cellular pattern drawing even on a single-cell level by closely scanning a microelectrode above the substrate and electrochemically producing the agent at the tip of the electrode. The present microelectrochemical cell patterning is applicable even for a previously cell-patterned substrate and for a grooved substrate. These unique technical features will have impacts on a variety of cell-based studies that require the analysis of heterotypic cell-cell interactions and cellular arrangement on an uneven surface such as semiconductor devices.     

Engineering Substrate Topography at the Micro‐ and Nanoscale to Control Cell Function

The interaction of mammalian cells with nanoscale topography has proven to be an important signaling modality in controlling cell function. Naturally occurring nanotopographic structures within the extracellular matrix present surrounding cells with mechanotransductive cues that influence local migration, cell polarization, and other functions. Synthetically nanofabricated topography can also influence cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization. We review the use of in vitro synthetic cell–nanotopography interactions to control cell behavior and influence complex cellular processes, including stem‐cell differentiation and tissue organization. Future challenges and opportunities in cell–nanotopography engineering are also discussed, including the elucidation of mechanisms and applications in tissue engineering.