Facilitating the culture of mammalian nerve cells with polyelectrolyte multilayers

When neuron-like cells (NLCs) derived from pluripotent embryonal carcinoma cells (P19) are cultured on bare tissue culture substrates, they require a monolayer of fibroblast cells to exhibit normal neurite outgrowth, behavior typical of neuronal cultures. However, substrate treatment with polyelectrolyte multilayers (PEMs) composed of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonic acid) (PSS) significantly improved these cultures. Cell morphology was more spread, indicative of healthy cells, and direct attachment of neuronal cell bodies to the treated surface was observed. Neuronal outgrowth across the surface was not dependent on an underlying fibroblast monolayer with the PEMs surface treatment. Additionally, the PEMs surface treatment can be used to condition various surfaces, facilitating neuronal cultures on surfaces which are natively hydrophilic (tissue culture polystyrene) or hydrophobic (poly(dimethylsiloxane), PDMS). Microfluidic networks were used to micropattern the PEMs onto PDMS, resulting in confined regions of cellular attachment and directed neuronal outgrowth. The ability of PEMs to encourage NLC attachment without supporting cells to a variety of surfaces and surface geometries greatly simplifies neuronal culture methodology and enables neuronal investigations in new environments.     

Calcium-Dependent Translocation of S100B Is Facilitated by Neurocalcin Delta

S100B is a calcium-binding protein that governs calcium-mediated responses in a variety of cells—especially neuronal and glial cells. It is also extensively investigated as a potential biomarker for several disease conditions, especially neurodegenerative ones. In order to establish S100B as a viable pharmaceutical target, it is critical to understand its mechanistic role in signaling pathways and its interacting partners. In this report, we provide evidence to support a calcium-regulated interaction between S100B and the neuronal calcium sensor protein, neurocalcin delta both in vitro and in living cells. Membrane overlay assays were used to test the interaction between purified proteins in vitro and bimolecular fluorescence complementation assays, for interactions in living cells. Added calcium is essential for interaction in vitro; however, in living cells, calcium elevation causes translocation of the NCALD-S100B complex to the membrane-rich, perinuclear trans-Golgi network in COS7 cells, suggesting that the response is independent of specialized structures/molecules found in neuronal/glial cells. Similar results are also observed with hippocalcin, a closely related paralog; however, the interaction appears less robust in vitro. The N-terminal region of NCALD and HPCA appear to be critical for interaction with S100B based on in vitro experiments. The possible physiological significance of this interaction is discussed.     

Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces

In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin was monitored by Quartz Crystal Microbalance with Dissipation (QCM-D) at two different concentrations, 20 μg/ml and 200 μg/ml, and the fibronectin adsorption experiments were complemented with antibody measurements. The QCM-D results show that the surface mass uptake is largest on the Au surfaces, while the number of polyclonal and monoclonal antibodies directed against the cell-binding domain (CB-domain) on the fibronectin (Fn) is significantly larger on the (HA) surfaces. Moreover, a higher number of antibodies bound to the fibronectin coatings formed from the highest bulk fibronection concentration. In subsequent cell studies with hMSC's we studied the cell spreading, cytoskeletal organization and cell morphology on the respective surfaces. When the cells were adsorbed on the uncoated substrates, a diffuse cell actin cytoskeleton was revealed, and the cells had a highly elongated shape. On the fibronectin coated surfaces the cells adapted to a more polygonal shape with a well-defined actin cytoskeleton, while a larger cell area and roundness values were observed for cells cultured on the coated surfaces. Among the coated surfaces a slightly larger cell area and roundness values was observed on HA as compared to Au. Moreover, the results revealed that the morphology of cells cultured on fibronectin coated HA surfaces were less irregular. In summary we find that fibronectin adsorbs in a more activated state on the HA surfaces, resulting in a slightly different cellular response as compared to the fibronectin coated Au surfaces.     

Distribution of a major surface-associated glycoprotein, fibronectin, in cultures of adherent cells

Fibronectin was present in media and cell layers of cultures of adherent cells from human skin, kidney, lung, chest wall, liver, and heart. Cell-surface fibronectin, visualized by immunofluorescence, was in dense fibrillar (cultures from lung), discrete fibrillar (e.g., cultures from skin), or punctate (some cultures from kidney) structures. The subunit sizes of cell-surface fibronectin and fibronectin soluble in medium appeared identical in sodium dodecyl sulfate-polyacrylamide gels. To explain the polymorphism of cell-surface fibronectin, there must be chemical differences among the fibronectins synthesized by different cell strains or factors in the cell layer which influence fibronectin binding and aggregation.     

Streptococcus mutans and Streptococcus intermedius adhesion to fibronectin films are oppositely influenced by ionic strength

Bacterial adhesion to protein-coated surfaces is mediated by an interplay of specific and nonspecific interactions. Although nonspecific interactions are ubiquitously present, little is known about the physicochemical mechanisms of specific interactions. The aim of this paper is to determine the influence of ionic strength on the adhesion of two streptococcal strains to fibronectin films. Streptococcus mutans LT11 and Streptococcus intermedius NCTC11324 both possess antigen I/II with the ability to bind fibronectin from solution, but S. intermedius binds approximately 20x less fibronectin than does the S. mutans strain under identical conditions. Both strains as well as fibronectin films are negatively charged in low ionic strength phosphate buffered saline (PBS, 10x diluted), but bacteria appear uncharged in high ionic strength PBS. Physicochemical modeling on the basis of overall cell surface properties (cell surface hydrophobicity and zeta potentials) demonstrates that both strains should favor adhesion to fibronectin films in a high ionic strength environment as compared to in a low ionic strength environment, where electrostatic repulsion between equally charged surfaces is dominant. Adhesion of S. intermedius to fibronectin films in a parallel plate flow chamber was completely in line with this modeling, while in addition atomic force microscopy (AFM) indicated stronger adhesion forces upon retraction between fibronectin-coated tips and the cell surfaces in high ionic strength PBS than in low ionic strength PBS. Thus, the dependence of the interaction on ionic strength is dominated by the overall negative charge on the interacting surfaces. Adhesion of S. mutans to fibronectin films, however, was completely at odds with theoretical modeling, and the strain adhered best in low ionic strength PBS. Moreover, AFM indicated weaker repulsive forces upon approach between fibronectin-coated tips and the cell surfaces in low ionic strength PBS than in high ionic strength PBS. This indicated that the dependence of the interaction on ionic strength is dominated by electrostatic attraction between oppositely charged, localized domains on the interacting surfaces, despite their overall negative charge. In summary, this study shows that physicochemical modeling of bacterial adhesion to protein-coated surfaces is only valid provided the number of specific interaction sites on the cell surfaces is low, such as on S. intermedius NCTC11324. Nonspecific interactions are dominated by specific interactions if the number of specific interaction sites is large, such as on S. mutans LT11. Its ionic strength dependence indicates that the specific interaction is electrostatic in nature and operative between oppositely charged domains on the interacting surfaces, despite the generally overall negatively charged character of the surfaces.     

Thermodynamic Investigation of Staphylococcus epidermidis interactions with protein-coated substrata

We evaluated self-assembled monolayers (SAMs) as potential coatings to prevent bacterial adhesion to biomaterials. Bacterial retention experiments were conducted on SAMs, some of which were coated with the model proteins fetal bovine serum (FBS) and fibronectin (FN). A thermodynamic approach was applied to calculate the Gibbs free energy changes of adhesion (DeltaG(adh)) of Staphylococcus epidermidis interacting with the substrates. When only nonspecific interactions controlled bacterial attachment, such as for the non-protein-coated substrates or the FBS substrates, the correlation between the thermodynamic predictions and measured values of bacterial retention was strong. However, when FN was adsorbed to the surfaces, the thermodynamic modeling underestimated bacterial adhesion, presumably since specific interactions between proteins of S. epidermidis and FN led to stronger attachment. Bacterial viability on the substrates was correlated with thermodynamic properties. For example, although bacteria attached more to surfaces having negative DeltaG(adh) values, these cells experienced the greatest loss of viability, presumably since strongly attached bacteria were unable to divide and grow. When the DeltaG(adh) values were decoupled into their components, we saw that acid-base interactions due to hydrogen bonding dominated the interactions of bacteria and proteins with each other and with the substrates in aqueous media. Finally, we discuss concerns regarding the use of the thermodynamic model to predict bacterial adhesion behavior in biomaterials systems.     

Effects of a serum spreading factor on growth and morphology of cells in serum-free medium

A heat-sensitive, trypsin-sensitive factor that promoted growth and spreading of cells in serum-free, hormone-supplemented medium was partially purified from human serum. The major portion of the proteins in these preparations migrated upon SDS-polyacrylamide gel electrophoresis with a mobility consistent with molecular weights between 60,000 and 90,000. The spreading activity, which we have termed serum spreading factor, stimulated growth and spreading of a wide variety of cell types. The serum spreading factor was similar to fibronectin in that it showed an affinity for the plastic cell culture substrate but was shown to be distinct from fibronectin by several criteria. This factor may prove useful in studies of cell attachment and spreading and in studies of the relationship of cell shape and cell proliferation.     

Fibronectin adsorption on titanium surfaces and its effect on osteoblast precursor cell attachment

The objective of this study was to investigate the adsorption of fibronectin on titanium (Ti) surfaces and the effect of pre-adsorbed fibronectin on osteoblast precursor cell attachment in vitro. Two different concentrations of bovine fibronectin were used in this study. Protein adsorption on Ti surfaces was analyzed using the micro bicinchoninic acid (BCA) protein assay. Cell concentration on Ti and fibronectin pre-adsorbed Ti surfaces after 3 h incubation was analyzed using the Vybrant™ cell adhesion assay. Cell morphology on Ti and fibronectin pre-adsorbed Ti surfaces was observed using scanning electron microscopy (SEM). After 180 min incubation, maximum adsorption of bovine fibronectin on Ti surfaces was observed. Fibronectin adsorption on Ti surfaces was observed to be significantly dependent on the initial concentration and the amount of incubation time. In the presence of 1 mg/ml fibronectin pre-adsorbed on Ti surfaces after 15 min, osteoblast precursor cell attachment on Ti surfaces was observed to be enhanced compared with control Ti surfaces, Ti surfaces pre-adsorbed with 1 mg/ml fibronectin for 180 min, and Ti surfaces pre-adsorbed with 0.1 mg/ml fibronectin for 15 and 180 min. No significant difference in cell attachment was observed between control Ti surfaces, Ti surfaces pre-adsorbed with fibronectin for 180 min, and Ti surfaces pre-adsorbed with 0.1 mg/ml fibronectin for 15 and 180 min. In addition, no differences in cell morphology of the attached osteoblast precursor cells on control Ti surfaces and Ti surfaces pre-adsorbed with fibronectin were observed in this study. It was concluded that an optimum concentration of adsorbed fibronectin on Ti surfaces plays an important role in governing cell attachment.     

Probing fibronectin-surface interactions: a multitechnique approach

The development of adhesive as well as antiadhesive surfaces is essential in various biomaterial applications. In this study, we have used a multidisciplinary approach that combines biological and physicochemical methods to progress in our understanding of cell-surface interactions. Four model surfaces have been used to investigate fibronectin (Fn) adsorption and the subsequent morphology and adhesion of preosteoblasts. Such experimental conditions lead us to distinguish between anti- and proadhesive substrata. Our results indicate that Fn is not able to induce cell adhesion on antiadhesive materials. On adhesive substrata, Fn did not increase the number of adherent cells but favored their spreading. This work also examined Fn-surface interactions using ELISA immunoassays, fluorescent labeling of Fn, and force spectroscopy with Fn-modified tips. The results provided clear evidence of the advantages and limitations of each technique. All of the techniques confirmed the important adsorption of Fn on proadhesive surfaces for cells. By contrast, antiadhesive substrata for cells avoided Fn adsorption. Furthermore, ELISA experiments enabled us to verify the accessibility of cell binding sites to adsorbed Fn molecules.     

Endothelial cell adhesion on polyelectrolyte multilayer films functionalised with fibronectin and collagen

The seeding of endothelial cells on biomaterial surfaces has become a major challenge to achieve better haemocompatibility of these surfaces. Multilayers of polyelectrolytes formed by the layerby-layer method are promising in this respect. In this study, the interactions of endothelial cells with multilayered polyelectrolytes films were investigated. The build-ups were prepared by selfassembled alternatively adsorbed polyanions and polycations functionalised with fibronectin and collagen. Anionic poly(sodium 4-styrenesulfonate) and cationic poly(allylamine hydrochloride) polyelectrolytes were chosen as a model system. Elaborated surfaces were characterised by electrochemical impedance spectroscopy and cyclic voltammetry. The modified electrode showed good reversible electrochemical properties and high stability in an electrolyte solution. The film ohmic resistance was highest when the film was coated with fibronectin; the parameters so determined were correlated with atomic force microscopy images. Cell colorimetric assay (WST-1) and immunofluorescence were used to quantify the cell viability and evaluate the adhesion properties. When cultured on a surface where proteins were deposited, cells adhered and proliferated better with fibronectin than with collagen. In addition, a high surface free energy was favourable to adhesion and proliferation (48.8 mJ m⁻² for fibronectin and 39.7 mJ m⁻² for collagen, respectively). Endothelial cells seeded on functionalised-polyelectrolyte multilayer films showed a good morphology and adhesion necessary for the development of a new endothelium.     

Cellular responses to patterned poly(acrylic acid) brushes

We use patterned poly(acrylic acid) (PAA) polymer brushes to explore the effects of surface chemistry and topography on cell-surface interactions. Most past studies of surface topography effects on cell adhesion have focused on patterned feature sizes that are larger than the dimensions of a cell, and PAA brushes have been characterized as cell repellent. Here we report cell adhesion studies for RBL mast cells incubated on PAA brush surfaces patterned with a variety of different feature sizes. We find that when patterned at subcellular dimensions on silicon surfaces, PAA brushes that are 30 or 15 nm thick facilitate cell adhesion. This appears to be mediated by fibronectin, which is secreted by the cells, adsorbing to the brushes and then engaging cell-surface integrins. The result is detectable accumulation of plasma membrane within the brushes, and this involves cytoskeletal remodeling at the cell-surface interface. By decreasing brush thickness, we find that PAA can be 'tuned' to promote cell adhesion with down-modulated membrane accumulation. We exemplify the utility of patterned PAA brush arrays for spatially controlling the activation of cells by modifying brushes with ligands that specifically engage IgE bound to high-affinity receptors on mast cells.     

Surface characterization of the extracellular matrix remaining after cell detachment from a thermoresponsive polymer

The temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) directly affects the attachment and detachment of cells cultured on these surfaces. At culture temperatures, cells behave similarly to those on tissue culture polystyrene (TCPS), while at room temperature, cells cultured on pNIPAM spontaneously detach as a confluent sheet. In comparison, cells grown on TCPS remain attached indefinitely after the temperature drop, requiring enzymatic or mechanical removal. In this work, we present an examination of the response of bovine aortic endothelial cells (BAECs) and extracellular matrix (ECM) proteins to plasma polymerized NIPAM (ppNIPAM) surfaces using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and immunostaining. Immunoassay results reveal that, although fibronectin, laminin, and collagen closely associate with the cell sheet, some collagen may be associated with the surface, as well. Our XPS results indicate that ppNIPAM surfaces after cell liftoff differ from their blank counterparts, the primary distinction being the presence of amide and alcohol species on ppNIPAM surfaces used for cell culture, possibly owing to the presence of a proteinaceous film. Finally, a comparison between ppNIPAM-treated surfaces used for cell culture versus control surfaces by principal component analysis of the ToF-SIMS data confirms that the surfaces differ; the presence of molecular ion fragments from amino acids (e.g., alanine, glycine, and proline) is the chief reason for this difference. Therefore, from our surface characterization of ppNIPAM-coated TCPS after cell liftoff, we conclude that although low-temperature liftoff of the BAEC monolayer is accompanied by the majority of the components of the ECM, some of the ECM proteins still remain at the surface.     

Concluding remarks and the challenge from the immune system

This meeting has covered a wide range of approaches to the study of interactions of biomembranes and one is struck by the progress in their analysis and the application of many new methods. The main theme of the meeting has been the structure of biomembranes and their components. Rather than pick highlights of these or attempt to summarise the findings in these Concluding remarks, I will instead summarise some of the interactions of the cells of the immune systems for which immunologists would like explanations at a molecular level. One of the features of the immune system is that it involves a variety of populations of cells that have complex migratory patterns and interactions that occur throughout life. The surfaces of these cells--the leukocytes--mediate interactions that are essential for the fine control of the immune system that ensures the rapid but controlled rejection of foreign materials such as viruses and bacteria. At the same time it must ensure that reactivity against self is prevented, otherwise autoimmune diseases such as rheumatoid arthritis and multiple sclerosis may result. Some of the features of the interactions involved are outlined in this short overview with more detailed analysis of the leukocyte cell surface given in ref. 1.     

Analysis of tubulin proteins and peptides in neuronal and non-neuronal tissues using immobilized pH gradients

The accurate identification of the individual protein products of multi-gene families is essential to the interpretation of data from a wide range of experimental approaches including molecular biology, protein chemistry, and cell biology. We have adapted immobilized pH gradient isoelectric focusing to provide high resolution of tubulin proteins. Here we use these techniques to investigate the heterogeneity of tubulin in several neuronal and non-neuronal tissues to provide an accurate evaluation of isotubulin composition. Of the ten sources examined, the greatest number of isotubulins was found in whole adult brain. Tubulin isolated from either neonate human or rat brain consists predominantly of the more basic alpha and beta isotubulins found in adult brain. Cerebrum, cerebellum, medulla and caudate nucleus all contain the same large number of isotubulins as in whole brain, but in varying proportions. Liver, kidney and spleen isotubulin populations are all similar to each other and consist of a simpler distribution than any neuronal tissue examined. The majority of the tubulin protein in these non-neuronal tissues is composed of only the most basic alpha tubulins and intermediately-charged beta tubulins. No isotubulins were identified that were unique to these three non-neuronal tissues. Tubulin from neuroblastoma cells has an isotubulin distribution grossly similar to non-neuronal sources but additionally contains two basic beta isotubulins found in adult brain that are absent from non-neuronal tissues.     

Influence of 40 Hz and 100 Hz Vibration on SH-SY5Y Cells Growth and Differentiation—A Preliminary Study

(1) Background: A novel bioreactor platform of neuronal cell cultures using low-magnitude, low-frequency (LMLF) vibrational stimulation was designed to discover vibration influence and mimic the dynamic environment of the in vivo state. To better understand the impact of 40 Hz and 100 Hz vibration on cell differentiation, we join biotechnology and advanced medical technology to design the nano-vibration system. The influence of vibration on the development of nervous tissue on the selected cell line SH-SY5Y (experimental research model in Alzheimer’s and Parkinson’s) was investigated. (2) Methods: The vibration stimulation of cell differentiation and elongation of their neuritis were monitored. We measured how vibrations affect the morphology and differentiation of nerve cells in vitro. (3) Results: The highest average length of neurites was observed in response to the 40 Hz vibration on the collagen surface in the differentiating medium, but cells response did not increase with vibration frequency. Also, vibrations at a frequency of 40 Hz or 100 Hz did not affect the average density of neurites. 100 Hz vibration increased the neurites density significantly with time for cultures on collagen and non-collagen surfaces. The exposure of neuronal cells to 40 Hz and 100 Hz vibration enhanced cell differentiation. The 40 Hz vibration has the best impact on neuronal-like cell growth and differentiation. (4) Conclusions: The data demonstrated that exposure to neuronal cells to 40 Hz and 100 Hz vibration enhanced cell differentiation and proliferation. This positive impact of vibration can be used in tissue engineering and regenerative medicine. It is planned to optimize the processes and study its molecular mechanisms concerning carrying out the research.     

Stem cell therapy for Alzheimer's disease and related disorders: current status and future perspectives

Underlying cognitive declines in Alzheimer''s disease (AD) are the result of neuron and neuronal process losses due to a wide range of factors. To date, all efforts to develop therapies that target specific AD-related pathways have failed in late-stage human trials. As a result, an emerging consensus in the field is that treatment of AD patients with currently available drug candidates might come too late, likely as a result of significant neuronal loss in the brain. In this regard, cell-replacement therapies, such as human embryonic stem cell- or induced pluripotent stem cell-derived neural cells, hold potential for treating AD patients. With the advent of stem cell technologies and the ability to transform these cells into different types of central nervous system neurons and glial cells, some success in stem cell therapy has been reported in animal models of AD. However, many more steps remain before stem cell therapies will be clinically feasible for AD and related disorders in humans. In this review, we will discuss current research advances in AD pathogenesis and stem cell technologies; additionally, the potential challenges and strategies for using cell-based therapies for AD and related disorders will be discussed.     

MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue

The development of powerful analytical techniques for specific molecular characterization of neural cell types is of central relevance in neuroscience research for elucidating cellular functions in the central nervous system (CNS). This study examines the use of differential protein expression profiling of mammalian neural cells using direct analysis by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-MS analysis is rapid, sensitive, robust, and specific for large biomolecules in complex matrices. Here, we describe a newly developed and straightforward methodology for direct characterization of rodent CNS glial cells using MALDI-MS-based intact cell mass spectrometry (ICMS). This molecular phenotyping approach enables monitoring of cell growth stages, (stem) cell differentiation, as well as probing cellular responses towards different stimulations. Glial cells were separated into pure astroglial, microglial, and oligodendroglial cell cultures. The intact cell suspensions were then analyzed directly by MALDI-TOF-MS, resulting in characteristic mass spectra profiles that discriminated glial cell types using principal component analysis. Complementary proteomic experiments revealed the identity of these signature proteins that were predominantly expressed in the different glial cell types, including histone H4 for oligodendrocytes and S100-A10 for astrocytes. MALDI imaging MS was performed, and signature masses were employed as molecular tracers for prediction of oligodendroglial and astroglial localization in brain tissue. The different cell type specific protein distributions in tissue were validated using immunohistochemistry. ICMS of intact neuroglia is a simple and straightforward approach for characterization and discrimination of different cell types with molecular specificity.     

Use of biospecific interactions of collagen, fibronectin and their fragments in affinity chromatography.

Various aspects of the application of fibronectin-collagen biospecific interactions in affinity chromatography are described. A new biospecific method for one-stage isolation of collagen peptides containing fibronectin-binding sites is proposed. The alpha 1 CB7-peptide of type-I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45,000 relative molecular mass) of fibronectin. The method gives highly purified preparations of alpha 1 CB7-peptide. This peptide, as well as some other collagen molecular fragments (alpha-chains, beta-components, alpha 1 CB8-peptide), were immobilized on Sepharose, and the properties of such affinity adsorbents obtained were studied. Adsorbents with immobilized alpha-chains and alpha 1 CB7-peptide had a fibronectin-binding capacity 1.5-2.0 times higher than commercial gelatin-Sepharose. Large-scale production of highly purified fibronectin from human plasma, using affinity chromatography on immobilized individual alpha-chains of collagen, was developed.     

Patterned co-culture of primary hepatocytes and fibroblasts using polyelectrolyte multilayer templates

This paper describes the formation of patterned cell co-cultures using the layer-by-layer deposition of synthetic ionic polymers and without the aid of adhesive proteins/ligands such as collagen or fibronectin. In this study, we used synthetic polymers, namely poly(diallyldimethylammonium chloride) (PDAC) and sulfonated polystyrene (SPS) as the polycation and polyanion, respectively, to build the multilayer films. We formed SPS patterns on polyelectrolyte multilayer (PEM) surfaces either by microcontact printing PDAC onto SPS surfaces or vice-versa. To create patterned co-cultures on PEMs, we capitalize on the preferential attachment and spreading of primary hepatocytes on SPS as opposed to PDAC surfaces. In contrast, fibroblasts readily attached to both PDAC and SPS surfaces, and as a result, we were able to obtain patterned co-cultures of fibroblast and primary hepatocytes on synthetic PEM surfaces. We characterized the morphology and hepatic-specific functions of the patterned cell co-cultures with microscopy and biochemical assays. Our results suggest an alternative approach to fabricating controlled co-cultures with specified cell-cell and cell-surface interactions; this approach provides flexibility in designing cell-specific surfaces for tissue engineering applications.