Large area micropatterning of cells on polydimethylsiloxane surfaces

Background

Precise spatial control and patterning of cells is an important area of research with numerous applications in tissue engineering, as well as advancing an understanding of fundamental cellular processes. Poly (dimethyl siloxane) (PDMS) has long been used as a flexible, biocompatible substrate for cell culture with tunable mechanical characteristics. However, fabrication of suitable physico-chemical barriers for cells on PDMS substrates over large areas is still a challenge.

Results

Here, we present an improved technique which integrates photolithography and cell culture on PDMS substrates wherein the barriers to cell adhesion are formed using the photo-activated graft polymerization of polyethylene glycol diacrylate (PEG-DA). PDMS substrates with varying stiffness were prepared by varying the base to crosslinker ratio from 5:1 to 20:1. All substrates show controlled cell attachment confined to fibronectin coated PDMS microchannels with a resistance to non-specific adhesion provided by the covalently immobilized, hydrophilic PEG-DA.

Conclusions

Using photolithography, it is possible to form patterns of high resolution stable at 37°C over 2 weeks, and microstructural complexity over large areas of a few cm². As a robust and scalable patterning method, this technique showing homogenous and stable cell adhesion and growth over macroscales can bring microfabrication a step closer to mass production for biomedical applications.

     

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.     

Stable sol-gel microstructured and microfluidic networks for protein patterning

We demonstrate the formation of micropatterned sol-gel structures containing active proteins by patterning with polydimethylsiloxane (PDMS) microchannels. To transport sol solution efficiently into the hydrophobic PDMS microchannels, a hydrophilic-hydrophobic block copolymer was used to impart hydrophilicity to the PDMS microchannels. Poor adhesion of the micropatterned gel structure onto glass slides was improved by treating the glass surface with a polymeric substrate. To minimize cracks in the gel microstructure, hybrid matrices of interpenetrating organic and inorganic networks were prepared containing the reactive organic moieties polyvinylalcohol or polyvinylpyrrolidone. Retention of biochemical activity within the micropatterned gel was demonstrated by performing immunobinding assays with immobilized immunoglobulin G (IgG) antibody. The potential application of microfluidics technology to immobilized-enzyme biocatalysis was demonstrated using PDMS-patterned microchannels filled with trypsin-containing sol-gels. This work provides a foundation for the microfabrication of functional protein chips using sol-gel processes.     

Microfluidic device‐assisted etching of p‐HEMA for cell or protein patterning

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2‐hydroxyethyl methacrylate) (p‐HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p‐HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p‐HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p‐HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p‐HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243–248, 2018     

Patterning of cells on functionalized poly(dimethylsiloxane) surface prepared by hydrophobin and collagen modification

Controllable cell growth on poly(dimethylsiloxzne) (PDMS) surface is important for its potential applications in biodevices. Herein, we developed a fully biocompatible approach for patterning of cells on the PDMS surface by hydrophobin (HFBI) and collagen modification. HFBI and collagen were immobilized on the PDMS surface one after another by using copper grids as a mask. HFBI self-assembly on PDMS surface converted the PDMS surface from hydrophobic to hydrophilic, which facilitated the following immobilization of collagen. Collagen had admirable ability to support cell adhesion and growth. Consequently, the HFBI/collagen-modified PDMS surface could promote cell adhesion and growth. What is more, the native PDMS surface did not support cell adhesion and growth. Patterning of cells was achieved by directly culturing 293T cells (the human embryonic kidney cell line) on the PDMS surface patterned with HFBI/collagen. Further studies by means of gene transfection experiment in vitro showed that the patterned cells were of good bioactivities. Herein, the biocompatible preparation of cell patterns on the PDMS surface could be of many applications in biosensor device fabrication.     

Fabrication of a circular PDMS microchannel for constructing a three-dimensional endothelial cell layer

We describe a simple and efficient fabrication method for generating microfluidic channels with a circular cross-sectional geometry by exploiting the reflow phenomenon of a thick positive photoresist. Initial rectangular shaped positive photoresist micropatterns on a silicon wafer, which were fabricated by a conventional photolithography process, were converted into a half-circular shape by tuning the temperature to around 105 °C. Through optimization of the reflow conditions, we could obtain a perfect circular micropattern of the positive photoresist, and control the diameter in a range from 100 to 400 μm. The resultant convex half-circular photoresist was used as a template for fabricating a concave polydimethylsiloxane (PDMS) through a replica molding process, and a circular PDMS microchannel was produced by bonding two half-circular PDMS layers. A variety of channel dimensions and patterns can be easily prepared, including straight, S-curve, X-, Y-, and T-shapes to mimic an in vivo vascular network. To form an endothelial cell layer, we cultured primary human umbilical vein endothelial cells inside circular PDMS microchannels, and demonstrated successful cell adhesion, proliferation, and alignment along the channel.     

Fabrication of lab-on chip platforms by hot embossing and photo patterning

In this paper, we review the approaches developed in our laboratory to fabricate polymer-based microfluidic devices to suit a range of applications in bio- or chemical analysis. Thermoplastic materials such as polycarbonate (PC) and poly(methyl methacrylate) (PMMA) are used to fabricate microfluidic devices via hot embossing. To emboss microchannels, we use hard stamps fabricated in silicon or soft stamps molded on poly(dimethylsiloxane) (PDMS). Hard stamps are fabricated on silicon wafers through photolithography and deep reactive ion etching (DRIE). Soft stamps are fabricated by casting PDMS prepolymer on silicon molds. To enclose the fluidic channels, direct fusion bonding was found to produce the highest bond strength with minimal structural deformation. One-step photolithographic methods have also been explored to produce via photochemical patterning microfluidic structures in photocurable materials. We use the photocurable capabilities of a PDMS copolymer, which incorporates a methacrylate crosslinker. Microfluidic channels are produced via one step-photopatterning processes by crosslinking the prepolymer mixture through a photomask. The smaller feature size attainable was 100 microm. Structures with higher spatial resolution are fabricated through a photoimprinting process whereby a mold is pressed against the precured mixture during UV crosslinking exposure. The application of the fabricated fluidic devices in electrophoretic ion analysis is also presented.     

Influence of cell adhesion and spreading on impedance characteristics of cell-based sensors

Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.     

Probing cell structure by controlling the mechanical environment with cell–substrate interactions

Recent results demonstrate the exquisite sensitivity of cell morphology and structure to mechanical stimulation. Mechanical stimulation is often coupled with cell–substrate interactions that can, in turn, influence molecular response and determine cellular fates including apoptosis, proliferation, and differentiation. To understand these effects as they specifically relate to compressive mechanical stimulation and topographic control, we developed a microfabricated system to grow cells on polydimethylsiloxane (PDMS) microchannel surfaces where we maintained compression stimulation. We also probed cellular response following compressive mechanical stimulation to PDMS substrates of varying stiffness. In these instances, we examined cytoskeletal and morphologic changes in living cells attached to our substrate following the application of localized compressive stimulation. We found that the overall morphology and cell structure, including the actin cytoskeleton, oriented in the direction of the compressive strain applied and along the topographic microchannels. Furthermore by comparing topographic response to material stiffness, we found a 40% increase in cell area for cells cultured on the microchannels versus softer PDMS as well as a decreased cell area of 30% when using softer PDMS over unmodified PDMS. These findings have implications for research in a diversity of fields including cell–material interactions, mechanotransduction, and tissue engineering.     

Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications

Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates. The use of PDMS structures in microfluidics and biological applications is discussed. We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer. Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity. The latter is mostly limited by the mechanical stability of the replicated PDMS structures. As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells.     

Metallization and Biopatterning on Ultra-Flexible Substrates via Dextran Sacrificial Layers

Micro-patterning tools adopted from the semiconductor industry have mostly been optimized to pattern features onto rigid silicon and glass substrates, however, recently the need to pattern on soft substrates has been identified in simulating cellular environments or developing flexible biosensors. We present a simple method of introducing a variety of patterned materials and structures into ultra-flexible polydimethylsiloxane (PDMS) layers (elastic moduli down to 3 kPa) utilizing water-soluble dextran sacrificial thin films. Dextran films provided a stable template for photolithography, metal deposition, particle adsorption, and protein stamping. These materials and structures (including dextran itself) were then readily transferrable to an elastomer surface following PDMS (10 to 70∶1 base to crosslinker ratios) curing over the patterned dextran layer and after sacrificial etch of the dextran in water. We demonstrate that this simple and straightforward approach can controllably manipulate surface wetting and protein adsorption characteristics of PDMS, covalently link protein patterns for stable cell patterning, generate composite structures of epoxy or particles for study of cell mechanical response, and stably integrate certain metals with use of vinyl molecular adhesives. This method is compatible over the complete moduli range of PDMS, and potentially generalizable over a host of additional micro- and nano-structures and materials.     

Microfluidic cell culture and its application in high-throughput drug screening: cardiotoxicity assay for hERG channels

Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval and hence risk for life-threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, the authors demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. They validate their microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP), and polydimethylsiloxane (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG) and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. The authors demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. The authors further show that experimental procedures can be streamlined by using direct in-channel immunofluorescence staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with 5 different drugs suggests that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules.     

Computational Modeling Reveals that a Combination of Chemotaxis and Differential Adhesion Leads to Robust Cell Sorting during Tissue Patterning

Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.     

Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily

During morphogenesis, cell-cell association patterns are dynamically altered. We are interested in how cell adhesion molecules can regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells are switched from one subtype to another. Artificial perturbation of this switch results in blocking of their escape from the neural tube. Intracellular modulations of cadherin activity also seem to play a role in regulation of cell adhesion. We identified p120ctn as a regulator of cadherin function in carcinoma cells. With such regulators, cells may make a choice as to whether they should maintain stable cell contacts or disrupt their association. Finally, we found another type of cadherin-mediated cell patterning: Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity in Drosophila imaginal discs. Thus, the cadherin superfamily receptors control the patterning of cell assemblies through a variety of mechanisms.     

A nanoparticle-based immobilization assay for prion-kinetics study

A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM). The method, termed Bioimprint™, creates a permanent cell 'footprint' in a non-biohazardous Poly (dimethylsiloxane) (PDMS) polymer composite. The transfer of nanometer scale biological information is presented as an alternative imaging technique at a resolution beyond that of optical microscopy. By transferring cell topology into a rigid medium more suited for AFM imaging, many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint™ replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale.     

Is cell culture stressful? Effects of degradable and nondegradable culture surfaces on U937 cell function

In vitro cell culture has become one of the most widely used techniques in biological and health sciences research, with the most common culture supports being either tissue culture grade polystyrene (TCPS) or polydimethylsiloxane (PDMS). It has previously been shown that monocyte-derived macrophages (MDMs) respond to material surface chemistry, synthesizing and releasing degradative activities that could produce products, which alter the cell's response. In this study, functional parameters of differentiated U937 macrophage-like cells were compared when cultured on nondegradable standard control surfaces versus models of biomaterials (polycarbonate-based polyurethanes) used in the manufacture of medical devices previously shown to degrade and/or elicit pathways of inflammation. Although the influence of PDMS and TCPS on cell function is often underappreciated by investigators, both surfaces elicited enzyme markers of inflammation. Cells on TCPS had the highest intracellular and released esterase activities and protein levels. Cells on PDMS had the most released acid phosphatase activity and protein (P < 0.001), as well as de novo 57- and 59-kDa released proteins. The criteria for defining an activated cell phenotype become critically important when materials such as PDMS and TCPS are used as standard control surfaces whether in experiments for research in elucidating metabolic pathways or in screening drugs and materials for therapeutic uses.     

Tunable hydroxyapatite wettability: Effect on adhesion of biological molecules

Surface wettability modifications of biocompatible materials and wettability patterning are attractive methods for directed biological cells immobilization for tissue engineering, drug delivery, gene transfer, etc. Hydroxyapatite is known as an implantable biomimetic material and a substrate for effective adhesion of biological cells of various origins. Here we report the use of a low-energy electron irradiation to achieve tunable wettability of the hydroxyapatite in a wide range of contact angles, from 10° to 100°, with accuracy of ±3°. The incident electrons generate electron/hole pairs resulting in significant variation of the surface potential of the hydroxyapatite semiconductor and give rise to pronounced wettability modification. Tailoring the gradually varied wettability state in the hydroxyapatite nanoceramics enabled the differential binding of biological materials with different surface properties, such as bovine serum albumin (BSA) and deoxyribonucleic acid (DNA).     

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO₂ wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO₂ regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.     

Self-adhesive microculture system for extended live cell imaging

Abstract

Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging.