Poissonian Cellular Potts Models Reveal Nonequilibrium Kinetics of Cell Sorting.

Cellular Potts models are broadly applied across developmental biology and cancer research. We overcome limitations of the traditional approach, which reinterprets a modified Metropolis sampling as ad hoc dynamics, by introducing a physical timescale through Poissonian kinetics and by applying principles of stochastic thermodynamics to separate thermal and relaxation effects from athermal noise and nonconservative forces. Our method accurately describes cell-sorting dynamics in mouse-embryo development and identifies the distinct contributions of nonequilibrium processes, e.g., cell growth and active fluctuations.

Rare-earth-doped biological composites as in vivo shortwave infrared reporters.

The extension of in vivo optical imaging for disease screening and image-guided surgical interventions requires brightly emitting, tissue-specific materials that optically transmit through living tissue and can be imaged with portable systems that display data in real-time. Recent work suggests that a new window across the short-wavelength infrared region can improve in vivo imaging sensitivity over near infrared light. Here we report on the first evidence of multispectral, real-time short-wavelength infrared imaging offering anatomical resolution using brightly emitting rare-earth nanomaterials and demonstrate their applicability toward disease-targeted imaging. Inorganic-protein nanocomposites of rare-earth nanomaterials with human serum albumin facilitated systemic biodistribution of the rare-earth nanomaterials resulting in the increased accumulation and retention in tumour tissue that was visualized by the localized enhancement of infrared signal intensity. Our findings lay the groundwork for a new generation of versatile, biomedical nanomaterials that can advance disease monitoring based on a pioneering infrared imaging technique.

Engineering hepatocyte functional fate through growth factor dynamics: the role of cell morphologic priming.

We have reported previously that cellular stimulation induced by variable mechanochemical properties of the extracellular microenvironment can significantly alter liver-specific function in cultured hepatocytes (Semler et al., Biotech Bioeng 69:359-369, 2000). Cell activation via time-invariant presentation of biochemical growth factors was found to either enhance or repress cellular differentiation of cultured hepatocytes depending on the mechanical properties of the underlying substrate. In this work, we investigated the effects of dynamic growth factor stimulation on the cell growth and differentiation behavior of hepatocytes cultured on either compliant or rigid substrates. Specifically, hepatotrophic growth factors (epidermal and hepatocyte) were either temporally added or withdrawn from hepatocyte cultures on Matrigel that was crosslinked to yield differential degrees of mechanical compliance. We determined that the functional responsiveness of hepatocytes to fluctuations in GF stimulation is substrate specific but only in conditions in which the initial mechanochemical environment induced significant cell morphogenesis. Our studies indicate that in conditions under which hepatocytes adopted a "rounded" phenotype, they exhibited increased levels of differentiated function upon soluble stimulation and markedly decreased function upon the depletion of GF stimulation. In contrast, hepatocytes that assumed a "spread" phenotype exhibited slightly increased function upon the depletion of GF stimulation. By examining the functional responsiveness of hepatocytes of differential morphology to varied fluctuations in GF activation, insights into the ability of cell shape to "prime" hepatocyte behavior in dynamic microenvironments were elucidated. We report on the possibility of uncoupling and, thus, selectively manipulating, the concerted contributions of GF-induced cellular activation and substrate- and GF-induced cell morphogenesis toward induction of cell function.

Functional engineering of hepatocytes via heterocellular presentation of a homoadhesive molecule, E-cadherin.

Engineering functional activity of liver cell cultures requires the modulation of specific cell-cell interactions. We have investigated the quantitative role of systematically varied presentation of the cell-cell adhesion molecule, E-cadherin, on the differentiated function of cocultured parenchymal liver cells, hepatocytes. Specifically, we incorporated different proportions of E-cadherin transfected L-929 chaperone cells and untransfected chaperone cells, within cultures of primary rat hepatocytes on a collagen substrate. By using a strongly adhesive substrate that restricted cadherin-induced variations in cell spreading and growth-arresting chaperone cells, we could carefully isolate the potential role of cell-cell adhesion on cell differentiation. Using immunofluorescence microscopy, we confirmed that cadherins expressed at hepatocyte-hepatocyte contacts as well as hepatocyte-chaperone contacts were crossreactive. However, hepatocytes cocultured with cadherin-presenting chaperone cells had a 55-65% increase in longterm function over hepatocytes cocultured with control, nonpresenting chaperone cells. Notably, the cadherin-induced increase in function occurred over and above the basal, coculture-induced functional elevation. Further, we quantified the stoichiometric importance of cadherin contacts by comparing established markers of hepatocyte functional activity across a graded range of E-cadherin presentation. At low levels of cadherin-mediated contacts, the induction of differentiated function was weak, while high levels of contacts elicited a marked increase in function. Thus, hepatocyte biochemical functions (albumin and urea secretion) were biphasically governed by the degree of cadherin-based contacts presented during culture. Overall, our results demonstrate the unequivocal role of cell-cell adhesion molecules in hepatocyte functional engineering, through the graded use of cadherin presentation from functionally incompetent, heterotypic chaperone cells.

Substrate microtopography can enhance cell adhesive and migratory responsiveness to matrix ligand density.

The regulation of cell motility by ligand density on substrates with variable microtopography is not well understood. In this report, we studied the adhesion and motility behavior of HepG2 cells on microtextured poly(glycolic-co-lactic)acid (PGLA) copolymer substrates, whose surface bioactivity was differentially modified through the adsorption of 0-5.5 ng/cm(2) collagen. Microtextured PGLA substrates were fabricated as thin films with a uniform surface distribution of micropores of median size of 3.1 +/- 1.5 microm and three-dimensional root mean squared roughness of 0.253 microm. Even in the absence of collagen, cells on microtextured substrates responded to substrate topography by exhibiting a 200% increase in adhesion strength compared with untextured controls and ventral localization of the intracellular adhesion protein vinculin. Further enhancement in adhesion strength (420% over untextured, untreated substrates) was demonstrated with bioactivated, microtextured surfaces, indicating that cell adhesion responses to topography and surface ligand density were cooperative. Our motility studies of cells on untextured substrates adsorbed with different levels of collagen demonstrated that a classical biphasic relationship between the cell population averaged migration rate, mu, and the collagen ligand density was preserved. However, comparison of cell motility responses between untextured and microtextured substrates indicates that the motility versus ligand density curve shifted, such that equivalent levels of cell motility were achieved at lower ligand density on microtextured surfaces. Furthermore, the maximum mu values achieved on the microtextured substrates exceeded those on untextured substrates by twofold. Taken together, we show that the magnitude of subcellular scale microtexture of a polymer substrate can sensitize the cell motility responsiveness to substrate ligand concentration; we suggest that the underlying mechanisms involve alteration in the degree of cell-substrate adhesivity as well as changes in the nature of ligand-induced cell activation processes.

Leukocyte chemosensory migration on vascular prosthetic biomaterial is mediated by an integrin beta2 receptor chain.

The ability of adherent activated leukocytes to migrate on implanted prosthetic biomaterial surfaces may be an early rate-limiting step in eliminating periprosthetic infection. The goal of this study was to explore the molecular mechanism governing leukocyte migration on the implantable cardiovascular prosthetic biomaterial, expanded polytetrafluoroethylene (ePTFE), in response to stimulation by the soluble chemokine, N-formyl-methionyl-leucyl-phenylalanine (fMLP). We used a population level migration assay to study the migration of polymorphonuclear leukocytes (PMN) on ePTFE, overlaid by a gelatin/agar composite. A theoretical random walk model was applied to describe fMLP-induced PMN migration on ePTFE in terms of an objective random cell migration coefficient, mu. Our results show that following stimulation with 0-10(-7) M fMLP, the value of mu ranged from 5.43 x 10(-9) to 1.08 x 10(-7) cm2/s, with a maximum value obtained at 10(-8) M fMLP. We probed the expression levels of various beta2 integrin receptor subunits and their contribution to the migratory function of ePTFE-adherent PMN over a wide range of fMLP concentration. We found that the expression of the integrin beta-chain, CD18, was also maximized at 10(-8) M fMLP, along with only slight changes in the expression of integrin alpha-chains (CD11a,b,c). We report that treatment with antibodies against either beta or combined alpha chains, but not individual alpha chains, inhibited PMN attachment to ePTFE at 10(-8) M fMLP, suggesting the likely role of combined beta2 receptor subunits in early adhesion events following stimulation. However, treatment with only anti-CD18 significantly lowered PMN migration on ePTFE (mu = 5.98 x 10(-9) cm2/s), and this degree of inhibition was much greater than that elicited by the combined treatment with antibodies recognizing all possible alpha-chains. Overall, we conclude that migratory behavior of chemokinetically stimulated PMN on ePTFE is mediated by the integrin beta chain pool, and is only weakly regulated by the integrin alpha chain.

Mechanochemical manipulation of hepatocyte aggregation can selectively induce or repress liver-specific function.

Controlled activation of hepatocyte aggregation is critical to three-dimensional (3D) multicellular morphogenesis during native regeneration of liver as well as tissue reconstruction therapies. In this work, we quantify the stimulatory effects of two model hepatotrophic activators, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), on the aggregation kinetics and liver-specific function of hepatocytes cultured on organotypic substrates with differing mechanical resistivity. Substrate-specific morphogenesis of cultured hepatocytes is induced on a tissue basement membrane extract, Matrigel, formulated at two distinct levels of mechanical compliance (storage modulus G', at oscillatory shear rate 1 rad/s, was 34 Pa for basal Matrigel and 118 Pa for crosslinked Matrigel). Overall, we report that growth factor stimulation selectively promotes the kinetics of aggregation in the form of two-dimensional corded aggregates on basal Matrigel and three-dimensional spheroidal aggregates on crosslinked Matrigel. Our analysis also indicates that costimulation with EGF and HGF (20 ng/mL each) cooperatively maximizes the kinetics of aggregation in a substrate-specific manner. In addition, we show that the role of growth factor stimulation on hepatocyte function is sensitively governed by the mechanical compliance of the substrate. In particular, on matrices with high compliance, costimulatory aggregation is shown to elicit a marked increase in albumin secretion rate, whereas on matrices with low compliance aggregation results in effective functional repression to basal, unstimulated levels. Thus, our studies highlight a novel interplay of physicochemical parameters of the culture microenvironment, leading to selective enhancement or repression of differentiated functions of hepatocytes, in concert with the activation of cellular morphogenesis.

Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis.

While microporous biopolymer matrices are being widely tested as cell culture substrates in hepatic tissue engineering, the microstructural basis for their control of cell differentiation is not well understood. In this paper, we studied the role of void size of collagen foams in directing the induction of liver-specific differentiated morphology and secretory activities of cultured rat hepatocytes. Hepatocytes cultured on collagen foams with subcellular sized pore diameters of 10 microm assumed a compact, cuboidal cell morphology, rapidly achieving monolayer coverage, and secreted albumin at the rate of 40 +/- 8 pg/cell/d. Increasing the pore size to 18 microm elicited a distinctly spread cellular phenotype, with discontinuous surface coverage, and albumin secretion rates declined precipitiously to 3.6 +/- 0.8 pg/cell/d. However, when collagen foams with an even higher average void size of 82 microm were used, hepatocytes exhibited high degree of spreading within an extensive three-dimensional cellular network, and exhibited high albumin secretory activity (26 +/- 0.6 pg/cell/d). The effect of void geometry on cellular ultrastructral polarity was further analyzed for the three void size configurations employed. The distribution of the cell-cell adhesion protein, E-cadherin, was primarily restricted to cell-cell contacts on the 10 microm foams, but was found to be depolarized to all membrane regions in cells cultured on the 18 and 82 microm foams. Vinculin-enriched focal adhesions were found to be peripherally clustered on cells cultured on 10 microm foams, but were found to redistribute to the entire ventral surface of cells cultured on the 18 and 82 microm foams. Overall, we demonstrate the significance of designing pore sizes of highly adhesive substrates like collagen toward selective cell morphogenesis in 2-D or 3-D. Subcellular and supercellular ranges of pore size promote hepatocellular differentiation by limiting 2-D cell spreading or effecting 3-D intercellular contacts, while intermediate range of pore sizes repress differentiation by promoting 2-D cell spreading.

PEG-variant biomaterials as selectively adhesive protein templates: model surfaces for controlled cell adhesion and migration.

Our study focused on the role of poly(ethylene glycol) (PEG) in actively regulating the biological responsiveness of protein-adsorbed biomaterials. To this end, we designed PEG-variant biomaterials from a family of tyrosine/PEG-derived polycarbonates to present surfaces ranging from low to intermediate levels of PEG concentration, below the PEG level requisite for complete abolition of protein adsorption. We analyzed the effect of PEG concentration on the amount, conformation and bioactivity of an adsorbed model protein, fibronectin, and on the attachment, adhesion strength and motility of L929 fibroblasts. Our results demonstrate that low levels of PEG can regulate not only the extent but also the conformation and specific bioactivity of adsorbed fibronectin. As the PEG concentration was increased from 0 to 6 mol%, the amount of adsorbed fibronectin decreased linearly yet the fibronectin conformation was altered such that the overall bioactivity of adsorbed fibronectin was uncompromised. We report that the degree of cell attachment varied with PEG concentration in a manner similar to the dependence of fibronectin bioactivity on PEG. In contrast, the nature of cell adhesion strength dependence on PEG paralleled the pattern observed for fibronectin surface concentration. Our studies also indicated that the rate of cell migration was inversely correlated with PEG concentration over a narrow range of PEG concentration. Overall, these results highlight the striking ability of PEG-variant biomaterials to systematically regulate the behavior of adsorbed cell adhesion proteins and, consequently, effect cell functions.

Quantitative analysis of the regulation of leukocyte chemosensory migration by a vascular prosthetic biomaterial.

The need for improved, infection-resistant vascular biomaterials calls for more objective evaluation of the immune pathophysiology of implantable prosthetic materials. In this study we have developed a new strategy to quantitatively characterize population-averaged responses of immune cell migration on vascular prosthetic materials. This approach, incorporating a chemokinetically regulated "biomaterial-gel" sandwich configuration, was applied to quantify both random and directed modes of the chemosensory migration of human neutrophil leukocytes on expanded polytetrafluoroethylene (ePTFE). Our studies show that (a) microporous, synthetic materials like ePTFE suppress the basal rate of random cell migration relative to that reported on non-porous control surfaces; (b) stimulation with chemoattractant (formyl peptide) can significantly elevate rates of random and directed migration on ePTFE; and (c) protein conditioning of ePTFE with albumin or immunoglobulin G can differentially modulate the rates and relative proportion of random and directional components of leukocyte migration response to chemoattractant. This, to our knowledge, is the first objective quantitation of chemokinetically regulated cell migration on implantable prosthetic materials.

Substrate-adsorbed collagen and cell secreted fibronectin concertedly induce cell migration on poly(lactide-glycolide) substrates.

Limited epithelial cell migration on synthetic polymeric biomaterials, such as polyesters, presents a serious challenge to their use as scaffolds for artificial skin analogs. The mechanisms by which a physiologic matrix interface on such polymers may regulate and promote cell migration under 'activated conditions' were the focus of this study. We have quantified the migration behavior of epidermal growth factor (EGF) stimulated epidermal keratinocytes on 50:50 poly-D,L(lactide-glycolide) (PLGA) substrates, following exogenous and cell-derived substrate conditioning based on the model matrix proteins, collagen and fibronectin. We report that 'non-conditioned' PLGA substrates elicited poor levels of keratinocyte migration. However, keratinocyte migration was significantly enhanced upon the adsorption of type I collagen, and was only weakly enhanced with fibronectin adsorption. Molecular analysis of the mechanism of enhanced migration on collagen-PLGA substrates showed that keratinocyte migration was sensitive to cell-derived fibronectin conditioning, but not to cell-secreted collagen conditioning. Fibronectin control of cell migration on collagen-PLGA was found to be both stoichiometric and biologically specific, mediated via adhesion involving keratinocyte alpha v integrin receptors. Based on our results, we propose a unique paradigm for induction of cell migration on a non-physiologic synthetic polymer using concerted interactions between primary, polymer-instructed matrix remodeling and secondary, cell-derived matrix remodeling.

Polymer substrate topography actively regulates the multicellular organization and liver-specific functions of cultured hepatocytes.

This study examines the role of topography of porous synthetic polymer substrates in regulating the tissue-specific morphogenesis of cultured hepatocytes. Porous foams of amorphous 50/50 poly(D,L glycolic-co-lactic acid) (PGLA) with a wide range of controlled pore-size distributions ( approximately 1 to 100 microm) were used as culture model surfaces. We found that the induction of microporosity in PGLA substrates significantly improved cell attachment and viability in comparison to those observed on non-porous PGLA films. A detailed evaluation of cellular morphogenesis on the microporous matrices showed that hepatocellular organization was sensitively dependent on the topographical feature size of the foam surfaces. Foams with subcellular size voids ( approximately 3 microm) induced kinetics of two-dimensional hepatocyte reorganization, yet limited the extent of three-dimensional aggregation. In contrast, foams with supercellular size voids ( approximately 67-microm) restricted hepatocyte motility, thereby promoting the kinetics of 3D aggregation. At intermediate void sizes ( approximately 17 microm), both 2D and 3D reorganization kinetics were promoted. Albumin secretory kinetics progressively increased on all void size configurations, the most rapid and sustained kinetics observed in supercellular sized voids, which may serve to minimize cell-polymer contacts and maximize cell-cell contacts in 3D. Overall, these studies demonstrate that void topography of porous polymer substrates is a critical textural feature to induce short-term cell adhesion and viability, and to also selectively regulate the kinetics and extent of multicellular spreading versus 3D aggregation. By virtue of its effects on cell adhesion and morphogenesis, the void topography of nonphysiological polymer scaffolds also is a powerful variable to microengineer hepatospecific activity of tissue analogs.

Flow-mediated cell stress induction in adherent leukocytes is accompanied by modulation of morphology and phagocytic function.

Leukocytes adherent to the surfaces of both vascular biomaterials and normal blood vessels experience blood flow induced shear stress. The goal of the reported studies was to investigate the effect of fluid flow on the morphology, phagocytic function and stress response induction in adherent immune cells. Shear approximating arterial, venous and intermediate levels were applied onto glass-adherent IC21 macrophages in a temperature-controlled parallel plate flow system. The results indicate that fluid flow induces a shear-dependent physiological stress response in adherent macrophages and that significant morphological changes accompany macrophage responses to shear stress. In addition, arterial flow conditions induce not only significant cell polarisation, but also enhanced phagocytic ingestion in glass-adherent IC21 macrophages. These findings suggest that blood flow induced shear stress may not only be consequent to adherent leukocyte activation, but may also be integral to the regulation of adherent leukocyte behaviour in vivo.

Leukocyte spreading behavior on vascular biomaterial surfaces: consequences of chemoattractant stimulation.

Chemoattractant-induced phenomena of polarity and migration of polymorphonuclear leukocytes (PMN) are believed to play a key physiological role in controlling bacterial infections on implantable vascular biomaterials. Our study targeted the spreading behavior of human PMN adherent to expanded polytetrafluoroethylene (ePTFE), pretreated with various plasma proteins, in response to the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMLP). To this end, a novel imaging configuration was developed to allow in situ reconstructive analysis of PMN 3-D morphology on opaque ePTFE surfaces, using optical sectioning confocal microscopy. Following fMLP stimulation, PMN morphological polarity was enhanced on all substrates studied except fibrinogen treated ePTFE. 3-D PMN morphometry revealed that in the absence of fMLP, overall cell spreading was minimized on albumin-treated ePTFE and maximized on fibrinogen and immunoglobulin-G-treated ePTFE. Following fMLP stimulation, overall PMN spreading increased markedly on untreated and albumin-coated ePTFE, while it stayed invariant on IgG and plasma treated ePTFE, and decreased on fibrinogen-treated ePTFE. Spatial analysis of PMN spreading following fMLP stimulation revealed enhanced PMN attachment on untreated and albumin treated ePTFE and diminished attachment on fibrinogen and plasma treated ePTFE. Thus, chemoattractant stimulation altered a wide range of PMN spreading attributes on ePTFE, including morphological polarity, substrate attachment, and 3-D membrane spreading, in a substrate dependent manner. These chemoattractant-induced spreading responses may also have important consequences for PMN phagocytosis. We report that fMLP stimulation led to enhanced unopsonized particulate phagocytosis on untreated and albumin treated ePTFE, but caused no discernible change in phagocytosis on other protein substrates. Thus, chemoattractant modulation of PMN spreading on ePTFE is highly substrate-regulated, and manifests in concerted effects on PMN phagocytosis.

Ultrasound of testicular epidermoid cysts.

The ultrasound findings of focal intratesticular lesions may sometimes be characteristic of benign pathology, which accounts for approximately 5% of all testicular tumours. Three cases of epidermoid cysts of the testis are presented along with a review of the literature. Recognition of the typical ultrasound appearances of this entity in a clinical situation of painless testicular swelling in the absence of a history of trauma and signs of inflammation can make testis-sparing surgery feasible.

Analysis of 3-D microstructure of porous poly(lactide-glycolide) matrices using confocal microscopy.

Porous matrices of biodegradable polymers are extensively used as scaffolds in tissue engineering and as drug delivery devices. A critical component of the design, processing, and utility of such polymeric systems concerns the local void microarchitecture. In this study, a novel approach based on confocal fluorescence imaging was employed to visualize and quantify in 3 dimensions (3-D) the individual and population-level void morphology within porous polymeric matrices. Poly(lactic acid-glycolic acid) copolymer matrices were cast to yield void configurations of variable void sizes but constant cumulative voidage. Using confocal microscopy, fluorescently saturated polymer matrices were optically sectioned into serial 2-D images, and 3-D void contours were reconstructed via object discrimination and connectivity analysis. The resultant data was used to quantitate the matrix microstructure and map its evolution following polymer degradation. Under conditions of accelerated degradation, matrix erosion was found to cause a significant change in the disposition of voids; this involves two processes (void formation and void enlargement), the extent of which was influenced by the initial void size and the duration of erosion. By virtue of providing both static and dynamic descriptions of the void morphology in poly(lactic acid-glycolic acid) matrices, this is the first spatiotemporal study of the 3-D microarchitecture of porous, bioerodible tissue analog matrices.

Analysis of surface microtopography of biodegradable polymer matrices using confocal reflection microscopy.

Currently, synthetic degradable polymers are frequently employed as culture substrates prior to cell transplantation and as implantable scaffolds for cellular infiltration during soft and hard tissue repair. The surface microstructure of matrices based on such polymers may be important in controlling cellular anchorage, spreading, and growth on the external surface, as well as infiltration into the voids of porous polymer scaffolds. While the chemistry, bulk structure, and mechanical properties of such polymers have been extensively studied, the surface microstructure has not yet been systematically examined, particularly following polymer degradation. In this study, we present the first account of the use of confocal laser-scanning reflection microscopy (CLSM) to visualize and quantitate the microtopography of the surface of porous matrices of poly(lactic acid)/poly(glycolic acid) (PLAGA) copolymers following polymer degradation. Utilizing this technique, we report that the surface morphology of PLAGA matrices changes significantly upon degradation, with increased local clustering of textured regions. Our quantitative analysis suggests that polymer degradation results in a lower spatially-averaged surface roughness, with significant cyclical variations observed at later time points. The computed surface correlation function was observed to increase upon degradation, confirming the results from our morphological studies. Finally, we demonstrate the efficacy of CLSM to concomitantly image both the polymer surface and locally attached cells, in real time.

Cell-cell interactions are essential for maintenance of hepatocyte function in collagen gel but not on matrigel.

The objective of this study was to examine the importance of cellular aggregation for the maintenance of liver-specific functions in hepatocytes. We used two culture matrix systems (collagen sandwich and Matrigel) to examine the responsiveness of albumin secretory function in cultured rat hepatocytes under various seeding conditions. With high cell seeding, both culture systems elicited comparable levels of elevated function. Under conditions of sparse seeding, however, their responses were quite distinct: collagen sandwiched cells exhibited a significant deterioration in secretion, while Matrigel-cultured cells retained their basal levels of function. This indicates that a critical degree of cell-cell interactions is essential for promoting function in the collagen sandwich, and in the Matrigel-cultured cells functions may be preserved by constitutive matrix-related phenomena, even in the absence of aggregation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 706-711, 1997.

Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration.

Extracellular matrix (ECM) geometry is an important modulator of cell polarity and function. For example, 3-dimensional matrices often more effectively induce differentiated cell function than traditional 2-dimensional substrates. The effect of ECM topology can be investigated in a controlled fashion using a technique whereby cells cultured on a single surface are overlaid with a second layer of ECM, thereby creating a "sandwich" configuration. Confluent monolayers of epithelial or endothelial cells overlaid in this fashion often reorganize into structures that are reminiscent of their native tissue. In the case of hepatocytes, the overlay causes a dramatic reorganization of the cytoskeleton, adoption of in vivo-like morphology and polarity, and expression of a wide array of liver-specific functions. In this short review, we use the sandwiched hepatocyte culture system to illustrate the effect of ECM geometry on cellular function. Pertinent studies are summarized in the context of defining the parallels, strengths, and limitations of this culture system as an in vitro model to study the physiology and morphogenesis of liver tissue. We also explore some of its potential uses as a model to study liver pharmacology and toxicology, and for the development of liver preservation techniques and liver-assist devices.