Hepatocellular phenotype in vitro is influenced by biophysical features of the collagenous substratum

Hepatocytes maintained on different substrata in vitro possess strikingly different morphological and biochemical features. Rounded, multicellular aggregates of hepatocytes are seen if the cells are plated onto Matrigel, a reconstituted basement membrane, whereas a flattened, monolayer of hepatocytes is observed with Vitrogen. Hepatocellular protein synthesis is much greater on the Matrigel, although collagen biosynthesis appears selectively enhanced on Vitrogen-grown hepatocytes. We determined that denatured type I collagen could be substituted for Matrigel as the substratum, with the hepatocytes remaining the same both morphologically and biochemically. This suggested that the cells respond to the biophysical state of the extracellular matrix not only to protein sequences that determine a binding site. Measurement of steady-state messenger RNA levels within cells cultured onto different matrices indicated that the fluid substrate of either Matrigel or denatured type I collagen were facilitative for induction of cytochrome P-450b/e, which was not seen with the rigid type I collagen substrata. In contrast the messenger RNA level for the cytoskeletal protein actin was decreased on the fluid matrices, suggesting that the rounded cells had a lower requirement for this protein. These findings indicate that hepatocytes are responsive to the biophysical state of the extracellular matrix, which can lead to significant changes in gene expression by the cells.     

Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue.     

Extracellular matrix-driven alveolar epithelial cell differentiation in vitro

During homeostasis and in response to injury, alveolar type II (AT2) cells serve as progenitor cells to proliferate, migrate, differentiate, and re-establish both alveolar type I (AT1) and AT2 cells into a functional alveolar epithelium. To understand specific changes in cell differentiation, we monitored morphological characteristics and cell-specific protein markers over time for isolated rat AT2 cells cultured on combinations of collagen, fibronectin and/or laminin-5 (Ln5). For all matrices tested, cultured AT2 cells displayed reduced expression of AT2 cell-specific markers from days 1 to 4 and increased expression of AT1-specific markers by day 3, with continued expression until at least day 5. Over days 5 to 7 in culture, cells took on an AT1-like phenotype (on collagen/fibronectin alone; collagen alone; or Ln5 alone), an AT2-like phenotype (on collagen/fibronectin/Ln5; or collagen/Ln5), or both AT1-like and AT2-like phenotypes (on collagen/fibronectin matrix with a subsaturating amount of Ln5). Cells transferred between matrices at day 4 of culture retained the ability to alter day 7 phenotype. We conclude that in vitro, (1) AT2 cells exhibited phenotype plasticity that included an intermediate cell type with both AT1 and AT2 cell characteristics independent of day 7 phenotype; (2) both collagen and Ln5 were needed to promote the development of an AT2-like phenotype at day 7; and (3) components of the extracellular matrix (ECM) contribute to phenotypic switching of alveolar cells in culture. The described tissue culture models provide accessible models for studying changes in alveolar epithelial cell physiology from AT2 cell progenitors to the establishment of alveolar epithelial monolayers that represent AT1-like, AT2-like, or a mix of AT1- and AT2-like cells.     

Effects of fibril- or fixed-collagen on matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1 production in the human hepatocyte cell line HLE

AIM: Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are central to the spontaneous resolution of liver fibrosis. The mechanisms involved have been investigated in hepatic stellate cells (HSC), but not in hepatocytes. We investigated the effects of fibril- and fixed-collagen on MMP-1 and TIMP-1 production in hepatocytes, using the HLE cell line. METHODS: Fibril type I and IV collagen were prepared by HCI digestion of type I and IV collagen, respectively. For fixed-collagen, culture dishes were coated with fibril type I or IV collagen and fixed by ultraviolet. Type I collagenase activity was measured using fluorescein isothiocyanate-labeled type I collagen. MMP-1 and TIMP-1 in HLE cells were measured by a one-step sandwich enzyme immunoassay. RESULTS: Both fibril type I and IV collagen significantly increased type I collagenase activity about two-fold compared with no fibril collagen. The effects of the fibril collagen were not affected by the coating condition. There was no significant difference in the effects on collagenase activity between cells cultured in medium containing fibril type I collagen and those cultured in the presence of type IV collagen. Both types of fibril collagen significantly increased MMP-1 production, and showed more than 10-fold higher levels of MMP-1 than the control. The enhanced MMP-1 production by fibril collagens was unaffected by the coating condition. By contrast, TIMP-1 production was not changed by the addition of fibril type I or IV collagen, and neither was it affected by the coating conditions. Coating with type I collagen significantly suppressed MMP-1 production by almost one-tenth compared with no coating. By contrast, TIMP-1 production was not affected by either the absence of a collagen coat or by increasing the concentration of the coating collagen. CONCLUSION: These results indicated that, in HLE cells, fibril- and fixed-collagen have opposite effects on MMP-1 production without affecting TIMP production. Fibril collagen induced collagenase activity by up-regulation of MMP-1 production without affecting TIMP-1 production. By contrast, fixed collagen reduced MMP-1 production. Our results suggest that hepatocytes might also play an important role in the regulation of the hepatic fibrosis alongside HSC.     

Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate

BACKGROUND: Liver injury transforms hepatic stellate cells into myofibroblast (MFB)-like cells. With recovery from injury, MFBs undergo apoptosis, but it is unknown whether they can also revert to quiescence.AIM: To determine whether human (h)MFBs become quiescent if cultured on a basement membrane-like substrate (Matrigel).METHODS: hMFBs obtained from cirrhotic liver were re-cultured on plastic or Matrigel. Expression of genes of collagen metabolism was assayed before and after transforming growth factor beta (TGFbeta) and Oncostatin M (OSM) stimulation.RESULTS: hMFBs had typical MFB-like morphology, with abundant alpha-smooth muscle actin (SMA) but no cytoplasmic lipid droplets. hMFBs re-cultured on Matrigel reverted to alphaSMA-negative, lipid droplet-positive quiescent morphology. alphaSMA, collagen alpha1(1) (COL1A1) and collagen alpha2(1) (COL1A2) messages were upregulated in hMFBs cultured on plastic, but suppressed by Matrigel. The opposite was true for metalloproteinase-1 mRNA. OSM but not TGFbeta reduced alphaSMA mRNA by 30% while TGFbeta but not OSM upregulated COL1A1 mRNA by 48%, in hMFBs on plastic. TGFbeta and OSM stimulated COL1A1 gene expression in Matrigel by 50 and 60%, respectively.CONCLUSIONS: Matrigel culture de-activates hMFBs yet collagen gene expression still responds to fibrogenic cytokines. The responses of hMFB gene expression to TGFbeta and OSM, are regulated differently by the extracellular matrix.     

Matrix Metalloproteinase-1 Activation via Plasmin Generated on Alveolar Epithelial Cell Surfaces

Plasmin is a potent protease related to tissue repair/remodeling not by fibrinolysis alone but also by activation of cytokines such as transforming growth factor and hepatocyte growth factor and by activation of matrix metalloproteases. We examined whether matrix matalloproteinase-1 was activated via plasminogen activation on surfaces of cultured alveolar epithelial cells (A-549). Cells were cultured overnight with plasminogen, pro-matrix metalloproteinase-1, and type I collagen as a substrate. Sodium dodecil sulfate–polyacrylamide gel electrophoresis was used to detect type I collagen degradation in culture supernatant. Collagen degradation corresponded to cell surface plasmin generation. No such finding was seen in the absence of cells or plasminogen. Alveolar epithelial plasminogen activation is important in matrix metalloproteinase-1 activation and thus presumably in tissue remodeling in pulmonary fibrosing pulmonary diseases such as idiopathic pulmonary fibrosis.     

胶原凝胶固定培养大鼠肝细胞的功能与形态观察

目的：探索混合胶原凝胶培养肝细胞的方法，观察培养鼠肝细胞的功能与形态特征。方法：两步法分离大鼠肝细胞，与I型鼠尾胶原溶液混合接种于培养瓶，待胶原液形成凝胶后，加培养液常规培养，观察培养鼠肝细胞的形态学特征和尿素合成及酶漏出量。结果：成功将大鼠肝细胞混合固定于胶原中，形成凝胶状进行培养。培养期间，始终能检测出鼠肝细胞合成分泌的尿素，而肝细胞乳酸脱氢酶漏出量较少，倒置相差显微镜下观察到典型的形态特征。结论：混合胶原凝胶培养方法能为肝细胞提供更接近体内的培养环境，可能适用于生物人工肝研究。     

Long-term production of major coagulation factors and inhibitors by primary human hepatocytes in vitro: perspectives for clinical application.

BACKGROUND/AIMS: Patients with coagulation factor disorders require lifelong symptomatic treatment. This is associated with limited efficacy and transmission risks. From a clinical point of view, hepatocyte transplantation offers a rational alternative but is currently being hampered by lack of functional stability of engrafted cells. It was the aim of our study to devise culture conditions providing stable cell polarity, attachment and growth factor stimulation to improve longevity and coagulation factor production. METHODS: Human hepatocytes (HC) were plated on different extracellular matrices, inside collagen gel or Matrigel. HC were grown inside growth factor-enriched serum-free medium (SFM) or exposed to media switching from differentiation (DM) to dedifferentiation (DeDM). RESULTS: Over more than 30 days in vitro human HC synthesized coagulation factors (factors VII, VIII, IX, fibrinogen) and coagulation inhibitors (antithrombin III, protein C). Protein synthesis was augmented when HC were grown inside a 3D collagen type I matrix, while Matrigel showed no additional benefit. Soluble growth factors improved coagulation factor production when applied in SFM or in sequential DM/DeDM. Coagulation factor levels ranged from 3% to 12% in the first week to 2.5-5% after 4 weeks, reaching biologically relevant levels. CONCLUSION: Preserved synthesis and secretion of coagulation factors in balanced proportion by human HC in this model may offer new perspectives for HC transplantation in coagulation defects of patients.     

Primary hepatocyte culture in collagen gel mixture and collagen sandwich

AIM: To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes. METHODS: A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined. RESULTS: Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration. Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phase-contrast microscope. There was little LDH-release during the culture period. CONCLUSION: Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivo environment, and are helpful for maintaining specific hepatic functions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the study of bioartificial livers.     

Culture of isolated bovine megakaryocytes on reconstituted basement membrane matrix leads to proplatelet process formation

We have enriched for bovine megakaryocytes and identified a culture system that may provide an in vitro model for platelet formation. Mature megakaryocytes with an unusually high ploidy distribution were obtained after differential centrifugation and velocity sedimentation of bone marrow cells through gradients of bovine serum albumin (BSA). The cell membranes of isolated megakaryocytes and megakaryocytes in vivo stained with antisera to human platelets and human platelet membrane GPIIIa. The microenvironment of bovine megakaryocytes in vivo was investigated using antibodies to types I and IV collagen and laminin. In an attempt to duplicate the microenvironment in vitro, bovine megakaryocytes were cultured on a reconstituted basement membrane matrix (Matrigel). The cells adhered to the gel, extended radial lamellipodia, and occasionally formed lengthy pseudopodia. Ultrastructural examination of these cells showed widening and coalescence of the megakaryocyte demarcation membranes (DMS), and inclusion of platelet granules, thin filaments, and microtubules in the processes. Very few DMS vesicles were present distally in the processes. The culture of megakaryocytes on a reconstituted basement membrane may closely model the in vivo megakaryocyte microenvironment and allow the study of thrombocytopoiesis in vitro.     

Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver.

Open fenestrations are a conspicuous feature of sinusoidal endothelial cells and allow free movement of plasma into the space of Disse. In hepatic fibrosis, the number of fenestrations decreases as interstitial collagen increases in the liver, a change that correlates with deposition of extracellular matrix in the space of Disse. In this study, the possibility of a causal relationship between altered fenestral morphology and perisinusoidal matrix has been examined by culturing rat sinusoidal endothelial cells on individual matrix proteins or on a native matrix consisting of human amniotic membrane with interstitial collagen (types I and III) on one side and basement membrane proteins (collagen types IV and V and laminin) on the other. Under culture conditions, individual components of the extracellular matrix failed to maintain fenestrations. A basement-membranelike gel matrix derived from the Engelbreth-Holm-Swarm tumor war similarly ineffective. Fenestral density and porosity (percentage of cell surface occupied by fenestrations) were significantly enhanced, however, when endothelial cells were cultured on the basement-membrane side of human amnion. These data suggest that support of endothelial fenestrations requires a complex matrix. In particular, physiologically derived basement membrane maintains fenestrations, whereas interstitial collagen matrix does not. The loss of fenestrations associated with hepatic fibrosis may be related in part to an accumulation of interstitial collagens in the space of Disse.     

Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains.

The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (> 90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human alpha 2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell layer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this collagen to be localized in an extracellular matrix surrounding the cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form of collagen has an unusual affinity for the cell layer in this system.     

Inhibition of angiogenesis on glycated collagen lattices

Summary Advanced glycation endproduct (AGE) accumulation in extracellular matrix proteins has been demonstrated in diabetic patients with a significant correlation with the severity of diabetic complications. AGE accumulation induces matrix protein cross-link formation, resulting in an increased stiffness of matrix fibres and the reduction of the susceptibility of matrix proteins to proteolytic degradation. We examined whether glycation-induced collagen cross-linking may affect vascular endothelial cell behaviours such as invasion, proliferation and differentiation, using the in vitro angiogenesis model of capillary-like structure formation in three-dimensional matrices of collagen type I. Endothelial cells cultured on collagen gel with angiogenic factors (the combination of fibroblast growth factor-2 and vascular endothelial growth factor) invaded the underlying collagen matrix, and organized capillary-like cord structures in the gel. We found that endothelial cell invasion into glycated collagen gel was significantly attenuated without any effect on proteinase activity including cell-associated plasminogen activator and matrix metalloproteinase in the conditioned medium. In addition, subsequent capillary-like cord formation was also inhibited in glycated collagen gel. In contrast, endothelial cell proliferation was enhanced on glycated collagen gel with or without angiogenic factors compared with control collagen gel. These results suggest that the structural alterations of extracellular matrix proteins through the glycation-induced cross-link formation affect the interaction between endothelial cell and extracellular matrix, resulting in the impairment of an adequate neovascularization in diabetic patients. [Diabetologia (1998) 41: 491–499] Received: 8 August 1997 and in revised form: 25 November 1997     

The characterization of normal and scleroderma skin fibroblasts cultured in a collagen gel matrix

Fibroblasts from normal and progressive systemic sclerosis involved skin more closely resemble in vivo cells when cultured in a collagen gel matrix, than do fibroblasts cultured on plastic. Ultrastructural studies show that the cytoskeleton and secretory organelles from these cells are better developed in the presence of a collagen gel. Fibronectin, glycosaminoglycans and collagen fibrils are produced by the cells and deposited in the preformed matrix. Collagen synthesis is greater in the scleroderma derived fibroblasts for all culture conditions with increased deposition in the matrix. Total collagen production form cells associated with the gel is less than that from those cultured on plastic, suggesting an inhibition of collagen synthesis by the preformed collagen matrix.     

Effects of extracellular matrix on the expression of peroxisomes in primary rat hepatocyte cultures

BACKGROUND/AIMS: Peroxisomes in wild-type cells vary between tissues and developmental stages. In the liver of some peroxisomal deficiency disorder patients, rare parenchymal cells express normal peroxisomes (mosaics); the mechanism is unknown. Our aim was to find factors regulating peroxisome expression. METHODS: Liver-specific as well as peroxisome characteristics were studied in three types of primary rat hepatocyte cultures. RESULTS: Total glutathione S-transferase activity and albumin secretion both increased in the collagen I sandwich and immobilization gel cultures. In contrast, in monolayers cultured on plastic, total glutathione S-transferase activity decreased and albumin secretion was only 30-40% compared to the collagen cultures. Glycogen rosettes typical of liver parenchymal cells were always abundant. Laminin and collagen IV-producing stellate cells were numerous in the monolayer but almost absent in the sandwich cultures. In 6-day-monolayer cultures, the number of liver-specific peroxisomes had decreased while atypical small or elongated peroxisomes appeared. Immunolabeling density for catalase and three beta-oxidation enzymes was decreased compared to adult rat liver; catalase specific activity in homogenates had dropped to 15% and 4% in the sandwich and monolayer cultures, respectively. In 17-day-sandwich cultures, some peroxisomes showed a very weak catalase reaction; total activity was 5%. Supplementation of the collagen type I cultures with several extracellular matrix factors could not prevent peroxisome dedifferentiation. CONCLUSION: The presence of these extracellular matrix components is not sufficient for normal peroxisome expression. It is suggested that hepatocyte-specific and peroxisomal features are regulated differently. The sandwich preserves hepatocyte differentiation better than the monolayer.     

Fibronectin regulates morphology, cell organization and gene expression of rat fetal hepatocytes in primary culture

BACKGROUND/AIMS: The extracellular matrix regulates hepatic development and regeneration, modulating the maintenance of liver architecture in the differentiated state. The aim of this work was to analyze how different extracellular matrix molecules modulate fetal hepatocyte morphology, growth and differentiation. METHODS: We cultured fetal hepatocytes either on plastic or on different extracellular matrix proteins, i.e., collagen I, fibronectin or E-C-L (entactin-collagen IV-laminin) and we analyzed cell attachment, morphological organization, proliferative response and gene expression. RESULTS: Cell attachment was increased by all the extracellular matrix proteins to a similar extent. However, only fibronectin facilitated the formation of elongated cord-like structures, reminiscent of liver plate organization. Immunocytochemical analysis of the cells in these structures revealed high levels of albumin and cytokeratin 18, phenotypical markers of parenchymal hepatocytes. Fibronectin did not block the mitogenic stimuli induced by epidermal growth factor in these cells and the elongated structures appeared either in the absence or in the presence of the mitogen. Cells cultured on fibronectin, regardless of whether epidermal growth factor was present or not, also presented the maximal levels of expression for liver specific genes, such as albumin or alpha-fetoprotein. This expression was coincident with an increased expression of hepatocyte nuclear factor (HNF)-4 and a higher HNF-1alpha/HNF-1beta ratio, when compared with those cells that were cultured on collagen or E-C-L extracellular matrix. CONCLUSIONS: These results suggest that fibronectin might play a differential role, as compared to other extracellular matrix proteins, in fetal hepatocyte organization and gene expression.     

生长因子及细胞外基质对肝前体细胞体外增殖及分化的影响

目的 明确多种生长因子对胚胎肝脏前体细胞体外增殖分化的影响。方法 用胶原酶从胎龄14．5d SD大鼠胚胎肝脏分离单个细胞，采用^3H-TdR掺入法检测生长因子对胎肝细胞体外增殖的促进作用，并观察生长因子及细胞外基质成分对胎肝干细胞集落形成的影响。采用免疫细胞双标记及G-6-P酶活性测定以检测肝前体细胞表面标记的表达及向成熟肝细胞的分化能力。结果 肝前体细胞在体外培养时显示克隆样生长的特性。促肝细胞生长因子(HGF)、表皮生长因子(EGF)能促进肝前体细胞的增殖，使DNA合成加速，并促进干细胞集落的形成及角蛋白19、白蛋白、G-6-P的表达。转化生长因子α对肝前体细胞的促增殖作用较弱。转化生长因子β对肝前体细胞的增殖起到抑制作用。细胞基质成分Ⅰ型胶原、Ⅳ型胶原、层黏连蛋白能促进肝干细胞集落的形成，而纤维连接蛋白的作用较弱。肝干细胞单克隆增殖需要生长因子及细胞外基质的共同参与，加入新鲜分离胎肝细胞培养液上清液时单细胞增殖较快，于第5天即形成细胞集落。结论 HGF、EGF对肝前体细胞的增殖分化起重要作用，细胞外基质成分亦参与了其增殖分化过程。肝干细胞单克隆培养除需生长因子和细胞外基质外，可能亦有某些造血细胞、间质细胞分泌的因子参与其增殖分化的调控。     

Types of collagen synthesized by normal rat liver hepatocytes in primary culture

Collagen formation is an important function of liver parenchymal cells that may be relevant to the pathogenesis of hepatic fibrosis. The types of collagen synthesized by cultured normal rat liver hepatocytes were examined. Cells isolated from adult rats by enzymatic dispersion of the liver were established in primary monolayer culture. Cells were then incubated with radiolabeled proline for 20 hr in the presence of ascorbate and the lathrogen beta-aminopropionitrile. Collagen secreted into the cell media was assessed separately from that in the cell layer. The greater proportion of newly synthesized collagen was associated with the cell layer. Collagen types were identified by ion exchange chromatography and by polyacrylamide gel electrophoresis. Types I, III, IV, and V collagen were present in both media and cell layer. Types III and V were the predominant types found. Very little Type I collagen was synthesized by these cultured normal hepatocytes. The percentages of Types I, III, IV, and V collagens, combining media and cell layer, were 6, 38, 19, and 36, respectively.