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 Specifically, hepatotrophic growth factors (epidermal and hepatocyte) were either temporally added or withdrawn from hepatocyte cultures on Matrigel that was crosslinked to yield differential degrees of mechanical compliance.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.     

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.     

Extended expression of liver functions of hepatocytes in collagen-contained cell aggregates (cell packs)

The functions of hepatocytes under the collagen-contained cell aggregate (cell pack) conditions were studied using liver-specific protein synthesis. Freshly isolated murine hepatocytes were suspended in the medium containing collagen and centrifuged, and the resultant cell masses were cultured on the porous membranes floating on the medium. In these cultures cells were attached to each other three-dimensionally with collagen present in the intercellular spaces. Cultured hepatocytes in the cell pack maintained high and stable activity in the expression of their functions for more than 2 weeks, even when cultured with the medium lacking any hormones and serum, whereas hepatocytes in monolayer cultures lost their functions within a week.Similarly, when the cell packs of rat hepatocytes were transplanted into rat spleens, they could retain viability in the form of cell aggregate with the expression of liver-specific albumin mRNA at a higher level than in the transplantated cell suspensions.The lifespan and the initial expression level of hepatocellular functions inculture were similar to that of the cell pack in cell aggregates without collagen and in cellular monolayers on the collagen gel respectively.It was concluded that the condition where cells are in contact witheach other has an important role in the expression of hepatocellular functions and collagen present in the intercellular spaces enhances the functional levels.     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Integrin binding and cell spreading on extracellular matrix act at different points in the cell cycle to promote hepatocyte growth.

This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes. Thus, these results suggest that ECM acts at two different points in the cell cycle to regulate hepatocyte growth: first, by activating the G0/G1 transition via integrin binding and second, by promoting the G1/S phase transition and switching off the default differentiation program through mechanisms related to cell spreading.     

Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA(+)FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA(+)FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues.     

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.     

原子力显微镜研究粘附分子与细胞膜上整合素分子的相互作用

目的：研究肝癌细胞弹性变化对其表达的整合素分子与配体分子相互作用的影响。方法：以壳聚糖/ 聚丙烯酰胺水凝胶作为 可变基底材料,并将人肝肿瘤细胞（HepG2）接种到不同软硬度壳聚糖/ 聚丙烯酰胺水凝胶基底上,利用原子力显微镜力与距离模 式定量测定不同软硬基底上生长的HepG2 肝瘤细胞膜表面整合素分子与层粘连蛋白分子之间相互作用力。结果：功能化的原子 力显微镜探针与不同软硬基底上生长的细胞所产生的粘附情况不相同,细胞生长在培养皿的为对照组;细胞生长在硬度为1000 Pa 壳聚糖/聚丙烯酰胺水凝胶基底上的为实验组,表达在HepG2 肝瘤细胞膜上的alpha-6-beta-1 整合素与其配体层粘连蛋白相互作用力 的大小分别为19± 7 pN和38.85± 19.7 pN。结论：基底软硬度会影响细胞整合素与配体分子间的相互作用。     

Size Distribution and Molecular Associations of Plasma Fibronectin and Fibronectin Crosslinked by Transglutaminase 2

Fibronectin (FN) is a ubiquitously expressed cell adhesion protein capable of assembling into large, extended fibrillar networks as part of an extracellular matrix (ECM) that regulates cell behavior. FN is a substrate for certain members of the transglutaminase family of protein-crosslinking enzymes-enzymes which can modify the ability of FN to support cell adhesion. In this study, we have analyzed the thermo-chemical stability of plasma FN in its noncrosslinked form, and after crosslinking by transglutaminase 2 (TG2), using dynamic light scattering. We report that FN is found in a generally globular (8.7 nm hydrodynamic radius), dimerized form in aqueous solutions, but unfolds into a linear arrangement at high ionic (1 M NaCl) and chaotropic (5 M urea) environments. FN conformation remained stable after multiple heating and cooling cycles ranging from 4 to 60 degrees C. Crosslinking of FN with TG2 formed large, multimeric complexes having high chemical stability in aqueous, high ionic and chaotropic environments, demonstrating that this covalent modification stabilizes FN. Given recent data that substrate (e.g. ECM) rigidity profoundly affects cell differentiation and behavior, we further studied how TG2 crosslinking affects the molecular rigidity of FN by obtaining atomic force microscopy nanoindentation measurements from untreated and crosslinked FN samples embedded in acrylamide gels. We demonstrate that TG2-mediated crosslinking of FN significantly increases Young's modulus (of elasticity), an observation of increased rigidity having important implications with respect to the biological role of ECM protein-crosslinking in cell signaling and guiding cell differentiation.     

Compliance of bio-adhesive substrates controls the kinetics of membrane-substrate association

Mechanical stiffness of bio-adhesive substrates is one of the major regulators of the cell adhesion and migration. In this study, we propose a theoretical model for the spontaneous growth of focal adhesion (FA) sites, on compliant elastic substrates, at the early stages of cellular adhesion. Using a purely thermodynamic approach, we demonstrate that the rate of membrane-substrate association decreases with increasing the compliance of the substrate. This can be considered as a reason for smaller spread area of the FA points after the stabilization of adhesion on compliant substrates, as reported by experiments. We also show that the extent to which the compliance of the substrate modulates the growth rate of adhesion site depends on the areal density of cell-adhesive ligands on the substrate.     

The role of actin filaments and microtubules in hepatocyte spheroid self-assembly

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.     

Co-culture system of hepatocytes and endothelial cells: two in vitro approaches for enhancing liver-specific functions of hepatocytes

Although hepatocyte transplantation and bioartificial liver support system provide new promising opportunities for those patients waiting for liver transplantation, hepatocytes are easily losing liver-specific functions by using the common in vitro cultured methods. The co-culture strategies with mimicking the in vivo microenvironment would facilitate the maintenance of liver-specific functions of hepatocytes. Considering that hepatocytes and endothelial cells (ECs) account for 80–90% of total cell populations in the liver, hepatocytes and ECs were directly co-cultured with hepatic stellate cells (HSCs) or adipose tissue-derived stem cells (ADSCs) at a ratio of 700:150:3 or 14:3:3 in the present study, and the liver-specific functions were carefully analyzed. Our results showed that the two co-culture systems presented the enhanced liver-specific functions through promoting secretion of urea and ALB and increasing the expressions of ALB, CYP3A4 and HNF4α, and the vessel-like structure in the co-culture system consisted of hepatocytes, ECs and ADSCs. Hence, our results suggested that the directly co-culture of hepatocytes and ECs with HSCs or ADSCs could significantly improve liver-specific functions of hepatocytes, and the co-culture system could further promote angiogenesis of ECs at a later stage. Therefore, this study provides potential interesting in vitro strategies for enhancing liver-specific functions of hepatocytes.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.     

Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes

Summary Some effects of culturing adult rat hepatocytes on each of four different substrates—laminin (LN), collagen type I (C-I), collagen type IV (C-IV), and fibronectin (FN)—have been investigated under defined conditions. No differential effect on the attachment of the cells to the various substrates was noted; however, the spreading of hepatocytes shortly after initial plating was most strikingly enhanced by FN, whereas LN exhibited little or no such enhancement. The two collagen substrates enhanced the spreading of hepatocytes more than did LN, but less than FN. The different substrates had no differential effect on the induction of tyrosine aminotransferase by dexamethasone and glucagon for at least the first 10 d in culture. The longevity of the hepatocytes was not changed significantly by any of the substrates, at least through the 14th d of culture. During the culture periods the hepatocytes at high cell density were maintained as confluent monolayers, regardless of the substrate on which they had been cultured. After 14 d of culture, γ-glutamyltranspeptidase activity was highest in cells cultured on C-IV, and lowest in those on FN. DNA synthesis in cultured hepatocytes at a low cell density was highest in cells cultured on FN, with decreasing levels of this parameter in cells cultured on C-IV, C-I, and LN, respectively. These results demonstrate that specific components of the extracellular matrix modulate both differentiated functions and the replication of hepatocytes cultured in serum-free medium. This work was supported in part by grants (CA-07175, CA-09135, CA-22484) from the National Cancer Institute, Bethesda MD. N. Sawada was supported by a Cancer Research Campaign Grant D (U.K.) from the International Union Against Cancer.     

Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing

We proposed a new molecular imprinting procedure based on molecular integration for the purpose of cell capture. We selected the cell-adhesive protein fibronectin (FN) as the imprinting protein for preparing templates and evaluated selective cell adhesion on the FN imprinting substrate. Silica beads with a diameter of 15 μm were used as the stamp matrix and FN molecules were adsorbed as a monolayer. The FN recognition sites were constructed by integrating a surfactant as the ligand and immobilizing it with new biocompatible photoreactive phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units. As control substrates, imprinting procedures were carried out using albumin (BSA imprinting substrate) and without imprinting protein (non-imprinting substrate). The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate, and induced the cell adhesion. On the other hand, on the non-imprinted and BSA imprinting substrates, the FN scarcely bound from the cell culture medium, and subsequent cell adhesion could not be observed on the substrate. These results indicate that the FN binding sites were well constructed by arranging the ligand surfactant to a suitable position and immobilized by the photoreactive MPC polymer. The MPC polymer prevented the nonspecific adsorption of proteins from the cell culture medium. We concluded that this procedure is convenient and can be potentially used for the preparation of surfaces for cell engineering devices.