Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography

In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.     

Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.     

Microfabrication-based modulation of embryonic stem cell differentiation

Embryonic stem (ES) cells form spontaneous aggregates during differentiation, and cell-cell communication in the aggregates plays an important role in differentiation. The development of a controlled differentiation scheme for ES cells has been hindered by the lack of a reliable method to produce uniform aggregate sizes. Conventional techniques, such as hanging drop and suspension cultures, do not allow precise control over size of ES cell aggregates. To surmount this problem, we microfabricated adhesive stencils to make mouse ES (mES) cell aggregates of specific sizes ranging from 100 microm to 500 microm in diameter. With this technique, we studied the effect of the initial aggregate size on ES cell differentiation. After 20 days of induction of differentiation, we analyzed the stem cell populations using gene and protein expression assays as well as biochemical functions. Notably, we found that germ layer differentiation depends on the initial size of the ES cell aggregate. Among the ES cell aggregate sizes tested, the aggregates with 300 microm diameter showed similar differentiation profiles of three germ layers as embryoid bodies made using the "hanging drop" technique. The smaller (100 microm) aggregates showed the increased expression of ectodermal markers compared to the larger (500 microm) aggregates, while the 500 microm aggregates showed the increased expression of mesodermal and endodermal markers compared to the 100 microm aggregates. These results indicate that the initial size of the aggregate is an important factor for ES cell differentiation, and can affect germ layer selection as well as the extent of differentiation.     

Microcontact printing of Concanavalin A and its effect on mammalian cell morphology

In this study a major lectin called Concanavalin A (ConA) has been micropatterned on a glass substrate by microcontact printing and the patterns have been characterized with fluorescent and atomic force microscope for their uniformity. Interaction of the patterns with mammalian cells has been investigated by culturing L929 mouse fibroblast cells on the ConA printed glass surface. Cell culture results obtained from the microcontact printed patterns have also been compared and benchmarked with another patterning technique named micromolding in capillaries (MIMIC). It has been revealed that in spite of molecular level heterogeneity and agglomeration of protein molecules in microcontact printed form, they can still interact with cell surface glycoproteins, impede the mobility of membrane receptor which results in altered morphology of the fibroblast cells.     

Manipulating microparticles with single surface-immobilized nanoparticles

This experimental study explores the capture and manipulation of micrometer-scale particles by single surface-immobilized nanoparticles. The nanoparticles, approximately 10 nm in diameter, are cationic and therefore attract the micrometer-scale silica particles in an analyte suspension. The supporting surface on which the nanoparticles reside is negative (also silica) and repulsive toward approaching microparticles. In the limit where there are as few as 9 nanoparticles per square micrometer of collector, it becomes possible to capture and hold micrometer-scale silica particles with single nanoparticles. The strong nanoparticle-microparticle attractions, their nanometer-scale protrusion forward of the supporting surface, and their controlled density on the supporting surface facilitate microparticle-surface contact occurring through a single nanoelement. This behavior differs from most particle-particle, cell-cell, or particle (or cell)-surface interactions that involve multiple ligand-receptor bonds or much larger contact areas. Despite the limited contact of microparticles with surface-immobilized nanoparticles, microparticles resist shear forces of 9 pN or more but can be released through an increase in the ionic strength. The ability of nanoparticles to reversibly trap and hold much larger targets has implications in materials self-assembly, cell capture, and sorting applications, whereas the single point of contact affords precision in particle manipulation.     

Micropatterning neuronal cells on polyelectrolyte multilayers

This paper describes an approach to adhere retinal cells on micropatterned polyelectrolyte multilayer (PEM) lines adsorbed on poly(dimethylsiloxane) (PDMS) surfaces using microfluidic networks. PEMs were patterned on flat, oxidized PDMS surfaces by sequentially flowing polyions through a microchannel network that was placed in contact with the PDMS surface. Polyethyleneimine (PEI) and poly(allylamine hydrochloride) (PAH) were the polyions used as the top layer cellular adhesion material. The microfluidic network was lifted off after the patterning was completed and retinal cells were seeded on the PEM/PDMS surfaces. The traditional practice of using blocking agents to prevent the adhesion of cells on unpatterned areas was avoided by allowing the PDMS surface to return to its uncharged state after the patterning was completed. The adhesion of rat retinal cells on the patterned PEMs was observed 5 h after seeding. Cell viability and morphology on the patterned PEMs were assayed. These materials proved to be nontoxic to the cells used in this study regardless of the number of stacked PEM layers. Phalloidin staining of the cytoskeleton revealed no apparent morphological differences in retinal cells compared with those plated on polystyrene or the larger regions of PEI and PAH; however, cells were relatively more elongated when cultured on the PEM lines. Cell-to-cell communication between cells on adjacent PEM lines was observed as interconnecting tubes containing actin that were a few hundred nanometers in diameter and up to 55 microm in length. This approach provides a simple, fast, and inexpensive method of patterning cells onto micrometer-scale features.     

On-chip culture system for observation of isolated individual cells

To investigate the properties of isolated single cells with their environment, we developed the differential analysis method for single cells using an on-chip microculture system. The advantages of the system are, (i). continuous cultivation of a series of isolated single cells or a group of cells under contamination free conditions, (ii). continuous observation and comparison of those cells with 0.2 microm spatial resolution by a phase-contrast/fluorescent microscopy system with digital image processing. The core of the system is an n x n (n = 20-50) array of chambers, where each is 20-70 microm in diameter and 5-30 microm deep holes etched into a biotin-coated 0.17 mm thick glass slide. The biotin-coated glass slide is covered with the streptavidin coated cellulose semipermeable membrane, which is fixed on the surface of the glass slide by streptavidin-biotin attachment, separating those holes from the nutrient medium circulating through a 'cover chamber' above. A single cell or group of cells can thus be isolated from environment perfused with the same medium, and the medium in each chamber can be changed within the diffusion time (<1/30 s). In addition, the microchamber volumes of specific cells or cell groups can be controlled by the sizes of the chambers. By using this system we found that the length of isolated Escherichia coli increased at 0.06 microm min(-1) between cell divisions regardless of the chamber volume, and that the cell concentration reached 10(12) cells ml(-1) under contamination free conditions. The system is thus particularly useful for one cell level analysis because the direct descendants of single cells can be cultured and compared in the isolated microchambers, and the physical properties of the cells in each microchamber can be continuously observed and compared.     

Density control of poly(ethylene glycol) layer to regulate cellular attachment

A wide variety of cells usually integrate and respond to the microscale environment, such as soluble protein factors, extracellular matrix proteins, and contacts with neighboring cells. To gain insight into cellular microenvironment design, we investigated two-dimensional microarray formation of endothelial cells on a micropatterned poly(ethylene glycol) (PEG)-brushed surface, based on the relationship between PEG chain density and cellular attachment. The patterned substrates consisted of two regions: the PEG surface that acts as a cell-resistant layer and the exposed substrate surface that promotes protein or cell adsorption. A PEG-brushed layer was constructed on a gold substrate using PEG with a mercapto group at the end of the chain. The density of the PEG-brushed layer increased substantially with repetitive adsorption/rinse cycles of PEG on the gold substrate, allowing marked reduction of nonspecific protein adsorption. These repeated adsorption/rinse cycles were further regulated by using longer (5 kDa) and shorter (2 kDa) PEG to construct PEG layers with different chain density, and subsequent micropatterning was achieved by plasma etching through a micropatterned metal mask. The effects of PEG chain density on pattern formation of cell attachment were determined on micropatterning of endothelial cells. The results indicated that cell pattern formation was strongly dependent on the PEG chain density and on the extent of protein adsorption. Notably, a PEG chain density high enough to inhibit outgrowth of endothelial cells from the cell-adhering region in the horizontal direction could be obtained only by employing formation of a short filler layer of PEG in the preconstructed longer PEG-brushed layer, which prevented nonspecific protein adsorption almost completely. In this way, a completely micropatterned array of endothelial cells with long-term viability was obtained. This clearly indicated the importance of a short underbrushed PEG layer in minimizing nonspecific protein adsorption for long-term maintenance of the active cell pattern. The strategy for cell patterning presented here can be employed in tissue engineering to study cell-cell and cell-surface interactions. It is also applicable for high-throughput screening and clinical diagnostics, as well as interfacing cellular and microfabricated components of biomedical microsystems.     

Soft lithography: masters on demand

We report an ultra-rapid prototyping technique for forming microchannel networks for lab-on-a-chip applications, called masters on-demand. Channels are produced by replica molding on masters formed by laser printing on flexible copper printed circuit board (PCB) substrates. Masters of various designs and dimensions can be individually or mass produced in less than 10 minutes. Using this technique, we have fabricated channels as narrow as 100 microm with heights ranging between 9 microm and 70 microm. Multi-depth channel fabrication is also reported, using a two-step printing process. The functionality of devices formed in this manner is verified by performing in-channel electrophoretic separations and culture and analysis of primary mammalian cells.     

Micropatterns of spores displaying heterologous proteins

Micropatterns of Bacillus thuringiensis spores were generated by a combination of surface chemistry, microcontact printing, and spore surface display technique. The outermost layers of B. thuringiensis spores were engineered to present enhanced green fluorescent protein (EGFP), and the biospecific interaction between biotin and streptavidin was utilized to spatially direct the EGFP-presenting spores onto the micropatterned surfaces. The viability of the micropatterned spores was confirmed by the pattern generation of vegetative cells after the germination.     

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.     

Patterned hydrogel substrates for cell culture with electrohydrodynamic jet printing

Cells respond to and are directed by physiochemical cues in their microenvironment, including geometry and substrate stiffness. The development of substrates for cell culture with precisely controlled physiochemical characteristics has the potential to advance the understanding of cell biology considerably. In this communication, E-jet printing is introduced as a method for creating high-resolution protein patterns on substrates with controlled elasticity. It is the first application of E-jet printing on a soft surface. Protein spots as small as 5 μm in diameter on polyacrylamide are demonstrated. The patterned hydrogels are shown to support cell attachment and spreading. Polyacrylamide substrates patterned by E-jet printing may be applied to further the study of cellular mechanobiology.     

A chip-based platform for the in vitro generation of tissues in three-dimensional organization

We describe a multi-purpose platform for the three-dimensional cultivation of tissues. The device is composed of polymer chips featuring a microstructured area of 1-2 cm(2). The chip is constructed either as a grid of micro-containers measuring 120-300 x 300 x 300 microm (h x l x w), or as an array of round recesses (300 microm diameter, 300 microm deep). The micro-containers may be separately equipped with addressable 3D-micro-electrodes, which allow for electrical stimulation of excitable cells and on-site measurements of electrochemically accessible parameters. The system is applicable for the cultivation of high cell densities of up to 8 x 10(6) cells and, because of the rectangular grid layout, allows the automated microscopical analysis of cultivated cells. More than 1000 micro-containers enable the parallel analysis of different parameters under superfusion/perfusion conditions. Using different polymer chips in combination with various types of bioreactors we demonstrated the principal suitability of the chip-based bioreactor for tissue culture applications. Primary and established cell lines have been successfully cultivated and analysed for functional properties. When cells were cultured in non-perfused chips, over time a considerable degree of apoptosis could be observed indicating the need for an active perfusion. The system presented here has also been applied for the differentiation analysis of pluripotent embryonic stem cells and may be suitable for the analysis of the stem cell niche.     

Simplest method for creating micropatterned nanostructures on PDMS with UV light

The fabrication of micropatterned structures on PDMS is a critical step in soft lithography, microfluidics, and many other PDMS-based applications. To substitute traditional mold-casting methods, we develop a simple method to create micropatterned nanostructures on PDMS in one step. After exposing a flat PDMS surface to a UV pen lamp through a photomask (such as a TEM grid), micropatterned nanostructures can be formed readily on the PDMS surface. We also demonstrate that fabricated PDMS can be used for the microcontact printing of protein immunoglobulin (IgG) on solid surfaces. This method is probably the simplest method of creating micropatterned nanostructures on PDMS reported so far because it does not need casting, surface coating, or chemical reagents. Only a UV pen lamp and a photomask are required, and this method can be performed under ambient conditions without vacuum. We expect that this method will greatly benefit researchers who use PDMS regularly in various applications such as soft lithography and microfluidics.     

Single cell epitaxy by acoustic picolitre droplets

The capability to encapsulate single to few cells with micrometre precision, high viability, and controlled directionality via a nozzleless ejection technology using a gentle acoustic field would have great impact on tissue engineering, high throughput screening, and clinical diagnostics. We demonstrate encapsulation of single cells (or a few cells) ejected from an open pool in acoustic picolitre droplets. We have developed this technology for the specific purpose of printing cells in various biological fluids, including PBS and agarose hydrogels used in tissue engineering. We ejected various cell types, including mouse embryonic stem cells, fibroblasts, AML-12 hepatocytes, human Raji cells, and HL-1 cardiomyocytes encapsulated in acoustic picolitre droplets of around 37 microm in diameter at rates varying from 1 to 10,000 droplets per second. At such high throughput levels, we demonstrated cell viabilities of over 89.8% across various cell types. Moreover, this ejection method is readily adaptable to other biological applications, such as extracting data from single cells and generating large cell populations from single cells. The technique described in the current study may also be applied to investigate stem cell differentiation at the single cell level, to direct tissue printing, and to isolating pure RNA or DNA from a single cell at the picolitre level. Overall, the techniques described have the potential for widespread impact on many high-throughput testing applications in the biological and health sciences.     

Inkjet-printed thiol self-assembled monolayer structures on gold: quality control and microarray electrode fabrication

Laterally structured, self-assembled monolayers (SAMs) of different thiols (HS-R-X, R = (CH 2) 3-16, X = -CH 3, -COOH, -NH 2) on gold have been prepared by inkjet printing. The printer is a modified, low-cost desktop printer (Epson Stylus Photo R200), the ink is a 1 mM solution of the thiol in ethanol/glycerol (6:1). The quality of inkjet-printed large area SAMs obtained in this study is between that of a layer self-assembled from a thiol solution and that obtained by soft lithography, according to cyclic voltammetry, electrochemical impedance spectroscopy, scanning electrochemical microscopy (SECM), and polarization-modulated Fourier transform infrared reflection-absorption spectroscopy (PM IRRAS). For the first time, simultaneous printing of two different thiols in a single print job as an alternative to sequential printing and backfilling is demonstrated. The smallest structures consisting of conductive disks of 40 microm diameter were analyzed as single spots by SECM and as random array electrodes with different average disk-disk distance. Conductive band electrodes with variable bandwidth (300 microm to 1 cm) are presented, as well as a pH switchable band structure. As compared to stamping, inkjet printing allows for simultaneous multiple thiol printing in a single print job with the resolution limited only by the droplet size and the precision of the translation stage.     

A mesh microcontactor for 2-phase reactions

The design, fabrication and use of mesh microcontactor structures is described. These allow contact between immiscible fluid phases (liquid/liquid or gas/liquid) enabling mass transfer and reaction between and within the phases. The phases are not mixed and are removed separately for subsequent use or analysis. The structures were designed for kinetic studies on biphasic reactions with the ability to handle sequential samples without excessive sample dispersion. Mass transport conditions are defined by fluid layer depths of 100 microm and the volume for each phase contacting in the reaction region was selected at 100 microl as sufficient for a range of analytical techniques. Micromeshes with pore diameter, depth, and spacing each of approximately 5 microm were formed by electrodeposition of nickel onto substrates with defined photoresist layers, and released by etching a copper sub-layer. Reactor enclosures defining chambers on each side of the mesh were formed from milled glass and metal components. The assembled structures have been used in flow-through and static fluid modes. System function has been demonstrated for both liquid/liquid and gas/liquid reactions. Test chemistries selected for ease of optical monitoring were hydrolysis of colourless fluorescein diacetate in toluene with transfer as fluorescein anion to aqueous alkali, and oxygen absorption into aqueous alkaline pyrogallol solution generating a coloured product, purpurogallin.     

Immobilization of histidine-tagged recombinant proteins onto micropatterned surfaces for cell-based functional assays

This letter describes a method for preparing protein microarrays that allow the functional analysis of proteins at a cellular level. This method involves the utilization of recombinant proteins genetically engineered to carry a fusion tag that has an affinity for metal ions. A micropatterned alkanethiol monolayer was used to prepare a microarray having multiple spots with immobilized metal ions. The fusion protein was chelated to the spots under physiological conditions. The feasibility of the method was demonstrated by culturing neural stem cells on the microarray that displayed oligohistidine-tagged epidermal growth factor.     

Micropatterns of double-layered nanofiber scaffolds with dual functions of cell patterning and metabolite detection

This paper describes the development of multi-functional nanofiber scaffolds consisting of multiple layers of nanofiber scaffolds and nanofiber-incorporated poly(ethylene glycol) (PEG) hydrogels. As a proof-of-concept demonstration, we fabricated micropatterned polymeric nanofiber scaffolds that were capable of simultaneously generating cellular micropatterns within a biomimetic environment and detecting cellular metabolic products within well-defined microdomains. To achieve this goal, we designed nanofiber scaffolds with both vertical and lateral microdomains. Vertically heterogeneous structures that were responsible for multi-functionality were realized by preparing double-layered nanofiber scaffolds consisting of an antibody-immobilized bottom layer of nanofibers and an upper layer of bare polystyrene (PS) nanofibers by a two-step sequential electrospinning process. Photopatterning of poly(ethylene glycol) (PEG) hydrogel on the electrospun nanofibers produced laterally heterogeneous micropatterned nanofiber scaffolds made of hydrogel microwells filled with a nanofibrous region, which is capable of generating cell and protein micropatterns due to the different interactions that cells and proteins have with PEG hydrogels and nanofibers. When HepG2 cells were seeded into resultant nanofiber scaffolds, cells selectively adhered within the 200 μm × 200 μm PS fiber microdomain and formed 180.2 ± 6.7 μm spheroids after 5 days of culture in the upper layer. Furthermore, immobilized anti-albumin in the bottom layer detected albumin secreted by micropatterned HepG2 cells with higher sensitivity than flat PS substrates, demonstrating successful accomplishment of dual functions using micropatterned double-layered nanofiber scaffolds.     

Conical tungsten tips as substrates for the preparation of ultramicroelectrodes

Here we describe a simple method to prepare voltammetric microelectrodes using tungsten wires as a substrate. Tungsten wires have a high tensile modulus and enable the fabrication of electrodes that have small dimensions overall while retaining rigidity. In this work, 125 microm tungsten wires with a conical tip were employed. For the preparation of gold or platinum ultramicroelectrodes, commercial tungsten microelectrodes, completely insulated except at the tip, were used as substrates. Following removal of oxides from the exposed tungsten, platinum or gold was electroplated, yielding surfaces with an electroactive area of between 1 x 10-6 and 2 x 10-6 cm2. Carbon surfaces on the etched tip of tungsten microwires were prepared by coating with photoresist followed by pyrolysis. The entire electrode was then insulated with Epoxylite except the tip, yielding an exposed carbon surface with an area of around 4 x 10-6 to 6 x 10-6 cm2. All three types of ultramicroelectrodes fabricated on the tungsten wire had similar electrochemical behavior to electrodes fabricated from wires or fibers insulated with glass tubes.