Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich

Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well thein vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells.     

Primary culture of rat hepatocytes using membrane-supported collagen sandwich with hormone-free medium

Summary Primary culture of rat hepatocytes in hormone-free medium using membrane-supported collagen sandwich maintained their cellular morphology and expressed albumin secretion for about 3 weeks in vitro. It was reconfirmed that mimicking the cellular environment in vivo was effective for cellular maintenance.     

Modulation of alpha-fetoprotein, albumin and transferrin gene expression by cellular interactions and dexamethasone in cocultures of fetal rat hepatocytes

Fetal hepatocytes were cultured alone or in association with primitive biliary cells (RLEC) in the presence or absence of dexamethasone. Cell-cell contacts were established 3 h or five days after hepatocyte seeding and their effects on hepatocyte growth and functional activities were evaluated in the presence or absence of dexamethasone. Establishment of cellular interactions with RLEC in coculture decreased hepatocyte growth, while it stimulated production of alpha-fetoprotein, albumin and transferrin. Addition of dexamethasone to coculture inhibited alpha-fetoprotein secretion and maintained the synthesis rate of albumin and transferrin together with an additional inhibition of DNA synthesis. The levels of mRNAs corresponding to the three proteins were also measured. We observed that the levels of alpha-fetoprotein, albumin and transferrin secretion in cocultures maintained in the presence or absence of dexamethasone were well correlated with the relative amounts of their corresponding mRNAs. Consequently, it may be assumed that the primitive mechanism involved in the increased functional activity of fetal hepatocytes in coculture is of pretranslational origin. Furthermore, the present data provide evidence that heterotypic interactions and dexamethasone act as distinct modulators of growth and maturation of fetal rat hepatocytes.     

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.     

Expression of cell adhesion molecule and albumin genes in primary culture of rat hepatocytes

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.     

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.     

Intermittent addition of HGF and TGF-β1 in rat primary hepatocyte culture

The effects of hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-β) on two morphological states of hepatocytes in monolayer and spheroid cultures, were examined in terms of their mitogenic ability and albumin expression. In monolayer culture on collagen-coated dishes, the increase in DNA content in the presence of HGF was observed when HGF was added within two days of cell isolation, whereas no increase in DNA was observed when HGF was added four days of cell isolation. DNA content increased even after four days, when HGF was added intermittently. On the other hand, spheroid formation was promoted on Primaria® dishes in HGF-free culture, whereas it was inhibited following the addition of HGF. No increase in DNA content was observed in spheroid cultures even in the presence of HGF throughout the culture period. The albumin production ability rapidly decreased in monolayer culture, but the decline was attenuated following the addition of HGF during the course of culture. A high albumin production ability was maintained independent of HGF supplementation in spheroid culture. Both DNA content and albumin production decreased rapidly following the addition of TGF-β1 in monolayer culture, and this decline was also attenuated following the addition of HGF to the medium.     

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration.

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.     

Extended primary culture of human hepatocytes in a collagen gel sandwich system

Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.     

The importance of proline on long-term hepatocyte function in a collagen gel sandwich configuration: regulation of protein secretion

A serum-free culture system for primary hepatocytes which maintains stabel high-level hepatocyte function for prolonged periods in culture has been developed. Isolated rat primary hepatocytes were cultured in serum-free media between two layer of gelled collagen in a sandwich configuration which reinstates the cellular polarity necessary for long-term function in vitro. Thsee serum-free hepatocyte cultures maintained near physiological rates of albumin and transferrin secretion for a minimum of 26 days in culture. L-Proline was shown to be critical for both the approach to steady state and maximal level of protein secretion. Analysis of does-response data gave K(m) values of 2.9 and 1.7 mug/mL for albumin and transferrin secretion, respectively.     

Plasminogen activator/plasmin system regulates formation of the hepatocyte spheroids

The isolated rat hepatocytes inoculated onto the surface of positively charged culture dishes are anchored initially and then begin to migrate and aggregate gradually to form multicellular spheroids detached from the dish. We studied the roles of fibrinolytic factors in the spheroid formation. The fibrinolytic factors, tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), were increased in the course of spheroid formation. Then, we introduced fibrinolytic inhibitors into the spheroid cultures to determine functions of fibrinolytic factors. Plasmin inhibitor inhibited markedly the spheroid formation. Interestingly, the anti-plasmin antibody showed different effect depending on the timing of its administration. In summary, we demonstrated for the first time that induction of PAs and ensuing plasmin generation on the cell surface play important roles in hepatocyte spheroid formation, and that plasmin is involved in the different processes such as cell migration and cell detachment in the formation of hepatocyte spheroid.     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.     

Composition of culture medium is more important than co-culture with hepatic non-parenchymal cells in albumin production activity of primary rat hepatocytes,and the effect was enhanced by hepatocytes spheroid culture in collagen gel

Co-culture of primary rat hepatocytes with hepatic non-parenchymal cells or sinusoidal endothelial cells for albumin production activity as an index of liver-specific function was studied. The co-cultures were effective for the expression and maintenance of albumin production activity. However, the co-culture effect was not observed when we used a suitable culture medium, which had already been reported to be sufficient for albumin production activity. Albumin production of dispersed cells in collagen gel culture was higher than that of spheroid culture. In addition, albumin production of spheroids in collagen gel culture was higher than that of spheroid culture and dispersed cell collagen gel culture with a suitable culture medium. We found that culture medium composition was more important than co-culture for expression and maintenance of albumin production. Furthermore, we found that cell–cell interaction was effective for the expression of albumin production, but heterotypic cell–cell interaction was not necessary.     

Modulation of cell proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox

The antiproliferative effects of the iron chelator O-trensox and the ornithine-decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were characterized in the rat hepatoma cell line FAO, the rat liver epithelial cell line (RLEC) and the primary rat hepatocyte cultures stimulated by EGF. We observed that O-trensox and DFMO decreased cell viabilty and DNA replication in the three culture models. The cytostatic effect of O-trensox was correlated to a cytotoxicity, higher than for DFMO, and to a cell cycle arrest in G0/G1 or S phases. Moreover, O-trensox and DFMO decreased the intracellular concentration of spermidine in the three models without changing significantly the spermine level. We concluded that iron, but also polyamine depletion, decrease cell growth. However, the drop in cell proliferation obtained with O-trensox was stronger compared to DFMO effect. Altogether, our data provide insights that, in the three rat liver cell culture models, the cytostatic effect of the iron chelator O-trensox may be the addition of two mechanisms: iron and polyamine depletion.     

Formation of spheroids of adult rat hepatocytes on polylysine-coated surfaces and thier albumin production

Summary Adult rat hepatocytes in primary culture formed spheroidal aggregates on the surfaces precoated with more than 0.01 mg-polylysine/mL. Epidermal growth factor (EGF) improved the formation and the maintenance of spheroid. The albumin secretion of the spheroids formed was maintained about 2 weeks at a high level.     

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.     

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.     

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ～80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ～70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G₀ phase, but that when they formed monolayers, they progressed to the G₁ phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions. © 1993 Wiley-Liss, Inc.     

Hepatocyte polarity and the peroxisomal compartment: a comparative study

In search of factors that regulate the phenotype of the peroxisomal compartment in wild-type liver parenchymal cells, we compared hepatocyte polarity to peroxisome differentiation, using adult liver as the standard. Differentiation parameters were evaluated in a three-dimensional culture model (spheroid), in 'sandwich' and monolayer primary hepatocyte cultures, and in 15.5 and 18.5-day-old foetal rat liver.Peroxisomes, studied by immunohistochemistry, enzyme histochemistry, and catalase specific activity, were better differentiated depending on foetal age (day 18.5 > day 15.5) and culture type (spheroid > sandwich > monolayer). The hepatocyte polarity markers ATP-, ADP-, and AMP-hydrolysing activities were, in all models, mislocalized at the lateral plasma membrane, whereas in contrast the multidrug resistance-associated protein 2 (mrp2) antigen was always correctly immunolocalized at the apical membrane domain. In cultures, the correct secretion of fluorescein (mrp2-mediated) into bile canaliculi was observed. Bile canaliculi (branching, ultrastructure and immunolocalization of the tight-junction associated protein ZO-1), were better differentiated in 18.5 than in 15.5-day-old foetal liver and in spheroid > sandwich > monolayer cultures.Our results show a parallelism between changes of the peroxisomal compartment and bile canalicular structure together with mrp2-mediated secretory function. Distinct polarization characteristics do not necessarily change simultaneously, suggesting different regulatory mechanisms.     

A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes

We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.