Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.     

Long-term culture of functional hepatocytes on chemically modified collagen gels

For long-term maintenance of functional hepatocytes in primary culture, a new culture system with chemically modified type-I collagen gel was developed. Isolated hepatocytes spread as flat cells and rapidly lost their viability and functions when cultured on native collagen gel. In contrast, they survived for several weeks when cultured on collagen gels that had been modified by treatment with sodium-borohydride (NaBH₄) or by digestion with pepsin, which resulted in destruction of crosslinking of collagen fibers and marked decrease in meachanical strength of the gels. These long-lived cells were round and aggregated and maintained high levels of various differentiated liver functions including albumin secretion and activities of tyrosine aminotransferase and P450. Moreover on collagen gels modified by treatment with NaBH₄ or pepsin, the cell showed less DNA synthesis in response to mitogenic stimulation than cells cultures on gel containing native collagen. Interestingly, crosslinking of these chemically modified gels with D-ribose resulted in changes in various phenotypes of hepatocytes cultures on them including shape, longevity, and functions expressed when the cells were cultured on native collagen gel, suggesting that the effect of modification of the collagen gel is reversible. Thus the structure of collagen gels, probably due to the degree of crosslinking, seems to affect the morphology, maintenance of differentiated functions, and growth of primary cultured hepatocytes.     

Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.     

Three-dimensional co-culture of hepatocytes and stellate cells

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.     

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.     

Prolonged lidocaine metabolizing activity of primary hepatocytes with spheroid culture using polyurethane foam as a culture substratum

Primary rat hepatocytes formed spheroids in the pores of polyurethane foam (PUF) used as a culture substratum. The hepatocytes in monolayer and spheroid stationary culture converted lidocaine to monoethylglycinexylidide (MEGX) which was N-deethylation of lidocaine. The metabolic activity of the hepatocytes/spheroid stationary culture system was 1.5∼2.0-fold higher than that of monolayer culture for 10 days. The activity of albumin production and cell survival of hepatocytes in monolayer and spheroid cultures decrease due to lidocaine treatment dependend on the lidocaine concentration, but the activity and cell survival in PUF/spheroid stationary culture were maintained at a higher level than that in monolayer culture under the lidocaine treatment. We developed a device for an in vitro liver model, drug metabolism simulator (DMS), using a PUF/spheroid packed-bed module including 4.00 ± 0.68 × 10⁷ hepatocytes and analyzed pharmacokinetics of lidocaine in a one-compartment model. Lidocaine clearance and extraction ratio of hepatocytes in the DMS corresponded to 1.354 ± 0.318 ml/min/g-liver and 0.677 ± 0.0159/g-liver, respectively (N=4). These values were comparable with in vivo values, 1.930 ml/min g-liver and 0.965/g-liver reported by Nyberg (1977). Consequently, PUF/spheroid culture maintained high lidocaine metabolizing activity over a long term and seems to provide a promising culture system as a drug metabolism simulator which will be used for drug screening, cytotoxicity tests and prediction of pharmacokinetics. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Spheroid formation and expression of liver specific functions of primary rat hepatocytes co-cultured with bone marrow cells

We have developed a new co-culture system consisting of adhesive bone marrow cells (A-BMCs), non-adhesive bone marrow cells (NA-BMCs) and hepatocytes with improved hepatocyte immobilization efficiency and better maintenance of liver specific functions. The composition of the inoculated cells affected the morphology of hepatocytes. Spheroids formed spontaneously when rat hepatocytes were co-cultured with bone marrow cells (BMCs). However, the addition of NA-BMCs to existing hepatocyte monolayers did not change their morphologies to spheroids. On the other hand, NA-BMCs dramatically increased hepatocyte immobilization efficiency. Hepatocytes co-cultured with either NA-BMCs or A-BMCs maintained their albumin production activities significantly better than hepatocyte culture alone. Interestingly, the two fractions of BMCs appear to have combination effects on hepatocytes to maintain albumin production activity. We conclude that co-culture of hepatocytes and BMCs is an effective strategy to enhance hepatocyte immobilization efficiency and functions in vitro.     

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

Influence of collagen gel substrata on certain biochemical activities of hepatocytes in primary culture

In order to study the influence of cell shape as modulated by the extracellular matrix on the cellular activity, hepatocytes isolated from liver were maintained on collagen I coated plastic substrata and collage I gel substrata and certain hepatocyte specific functions were investigated. The incorporation of³[H]-leucine into total proteins and albumin secreted by cells maintained on collagen gel was found to be significantly higher compared to those maintained on a collagen coated plastic substrata, indicating that hepatocytes on collagen gel have an enhanced albumin synthesizing capacity. Increased incorporation of³⁵[S]-sulphate into total proteoglycans (PG) and a relatively higher fraction of the³⁵[S]-PG in the extracellular space showed an increased rate of synthesis and secretion of sulphated PGs by cells maintained on collagen gels. But in contrast to the above results, the incorporation of³[H]-leucine into cytokeratins C₈, C₁₈ and actin were significantly low in cells maintained on collagen gel. The tyrosine amino transferase activity exhibited by hepatocytes preincubated with dexamethasone on collagen gel was also significantly low. The different forms of collagen substrata appeared to have no effect on the amino acid transport by hepatocytes, further suggesting that the various hepatocyte specific functions are not uniformly altered when hepatocytes are maintained on three-dimensional collagen gel substrata. These results indicate that the shape of the cell as determined by the nature of the matrix substratum influences the synthetic activity of secretory proteins and those remaining intracellularly, differently.     

Effects of degree of cell-cell contact on liver specific functions of rat primary hepatocytes

Cell-cell interaction and the extracellular matrix (ECM) are believed to play essential roles duringin vitro culturing of primary hepatocytes in the control of differentiation and in the maintenance of tissue specific functions. The objective of this study was to examine the effects of degree of cell-cell contact (DCC) on liver specific function of rat primary hepatocytes. Hepatocyte aggregates with various degrees of cell-cell contact,i.e., dispersed cells, longish aggregate, rugged aggregate, and smooth spheroid were obtained at 1, 5–6, 15–20, and 36–48 hrs, respectively in suspension cultures grown in spinner flasks embedded in Caalginate bead and collagen gel in order. The smooth spheroids displayed a decrease in viability and functional activities. This may result from mass transfer limitation and shear damage caused by agitation during aggregation. The rugged aggregate showed a higher viability and albumin secretion rate than the dispersed cells or the other aggregates. This result indicates the possible enhancement of a bioartificial liver's (BAL) performance using primary hepatocytes and the reduction in time to prepare a BAL through optimization of the immobilization time.     

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.     

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

The effect of two culture configurations (single collagen gel and double collagen gel) and of two hormones (insulin and glucagon) on the differentiated status and the intracellular nucleotide pools of primary porcine hepatocytes was investigated. The objective was to analyze and monitor the current state of differentiation supported by the two culture modes using intracellular nucleotide analysis. Specific intracellular nucleotide ratios, namely the nucleoside triphosphate (NTP) and the uridine (U) ratio were shown to consistently reflect the state of dedifferentiation status of the primary cells in culture affected by the presence of the two hormones insulin and glucagon. Continuous dedifferentiation of the cells was monitored in parallel by the reduction of the secretion of albumin, and changes in UDP-activated hexoses and UDP-glucuronate. The presence of insulin maintained the differentiated status of hepatocytes for more than 12 days when cultivated under double gel conditions whereas glucagon was less effective. In contrast, cells cultivated in a single gel matrix immediately started to dedifferentiate upon seeding. NTP and U ratios were shown to be more sensitive for monitoring dedifferentiation in culture than the albumin secretion. Their use allowed the generation of an easily applicable NTP–U plot in order to give a direct graphical representation of the current differentiation status of the cultured cells. Moreover, the transition from functional and differentiated hepatocytes to dedifferentiated fibroblasts could be determined earlier by the nucleotide ratios compared to the conventional method of monitoring the albumin secretion rate.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Albumin Synthesis by Rat Hepatocytes Cultured on Collagen Gels Is Sustained Specifically by Heparin

We investigated the influence of various kinds of glycosaminoglycans (GAGs) in collagen gels on the maintenance of albumin synthesis in primary culture of rat hepatocytes. Among the GAGs examined (heparin, heparan sulfate, keratan sulfate, chondroitin sulfate A, dermatan sulfate, and hyaluronic acid), only heparin-containing collagen gel cultures could significantly sustain albumin synthesis. However, other GAGs, such as heparan sulfate and keratan sulfate, had almost no effect on the maintenance of albumin synthesis. Heparin in collagen gels exhibited a dose-dependent effect on albumin synthesis: heparin at 400 μg/ml-collagen solution maintained albumin synthesis for over 3 weeks. On the other hand, when an equivalent amount of heparin was added directly to the collagen gel culture medium, it prolonged albumin synthesis for only 10 days. The results demonstrate that specific regulation of albumin synthesis by heparin was significantly promoted by coincubating it with collagen, suggesting that some specific interaction between heparin and collagen might be of importance for the maintenance of hepatocyte functions.     

Serum-free medium conditions for steroidogenesis of bovine follicular thecal cells cultured on collagen gel matrix

Summary Thecal cells isolated from bovine ovarian follicles were cultured with a serum-free basal medium or a serum-free complete medium in the presence or absence of collagen gel matrix, and their cellular proliferation and steroidogenesis were compared with those of cells cultured with a serum-containing medium. The cells cultured with the serum-free basal medium produced larger amounts of progesterone, androstenedione, and estradiol than the cells cultured with the serum-containing medium, but no appreciable cell proliferation was observed in the serum-free medium. Response of thecal cells to 8 bromo-cAMP, a steroidogenic agent, varied according to the type of steroid production examined and the type of culture medium used. In a cultivation period of 4 d, progesterone production was stimulated about five-fold by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium without collagen matrix, whereas androstenedione production was stimulated about three- to fourfold in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium with or without collagen matrix. Estradiol production, however, was significantly suppressed by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and also in the serum-containing medium. Thus, among the conditions examined, the most suitable primary culture media for steroidogenesis of thecal cells were the serum-free media, especially serum-free complete medium on collagen gel matrix.     

Effects of matrix components on aromatase activity in breast stromal cells in culture

Local estradiol production within breast tissue is maintained by the aromatase cytochrome P450_arom complex, which has been localized primarily to the stromal component of tumors but also has been detected in the breast epithelial cells. Paracrine interactions between stromal and epithelial components of the breast are critical to the sustained growth and progression of breast tumors. Maintenance of the differentiated state, including hormone and growth factor responsiveness, requires extracellular matrix proteins as substrata for cells. This research has focused on developing a cell culture system that more closely mimics in vivo interactions in order to dissect actual paracrine signaling between these two cell types. Human fibroblasts were isolated from breast tissue and were maintained in a cell culture system grown on plastic support or on a collagen I support matrix. The collagen I matrix model supports cell maintenance and subsequent differentiation on collagen rather than maximal proliferation, therefore allowing for a more accurate environment for the study of hormonal control and cellular communication. Initial experiments compared aromatase activity of patient fibroblasts grown on plastic versus collagen I using the tritiated water release method. Constitutive aromatase activity was found to be lower when cells were grown on a collagen gel for 4–7 days (7.7 fold lower) using DMEM/F12 containing 10% dextran coated charcoal stripped serum. However, fibroblasts grown on collagen I appeared to be significantly more responsive to stimulation by 100 nM dexamethasone (plastic: 6.0 fold induction, collagen: 33.2 fold induction) when pretreated for 12 h prior to measurement of aromatase activity. In an effort to examine paracrine interactions between the stromal and epithelial cells in breast tissue, experiments using conditioned media from fibroblast cultures were performed. Testosterone administration to fibroblasts results in the production of estradiol into the media in sufficient concentrations to elicit an increase in pS2 expression when the conditioned media is administered to MCF-7 cells. The addition of a potent aromatase inhibitor resulted in a complete suppression of fibroblast-derived estrogens and showed only a modest increase in pS2 expression. Culturing breast fibroblasts and epithelial cells on extracellular matrix allows for a more meaningful examination of the paracrine interactions between these cell types within the context of an appropriate extracellular environment. This study highlights the need for evaluation of gene expression in cell culture systems that accurately reflect the tissue microenvironment.     

Long-term culture of hepatocytes from human adults

A long-term primary human hepatocyte culture retraining liver-specific functions is important and essential for basic research and for the future development of hepatocyte-based applications. We established a normal hepatocyte culture system from excess normal tissues obtained from adult liver cancer patients who received partial liver resection. Hepatocytes were isolated after perfusion and enzymatic disaggregation, and were first maintained in hormonally defined media on a Matrigel matrix, and then transferred to collagen sandwich gel. The hepatocytes formed clusters on the Matrigel matrix and increased in size and numbers with time of culture and eventually grew into spheroids of variable sizes. After being transferred to collagen gel, the cells migrated outward from spheroids to form a monolayer with cuboidal or polygonal cell shapes with granular cytoplasm and continued to proliferate. Cellular functions specific for hepatocytes were analyzed using immunoblot assay for proteins specifically secreted by the liver cells on different days of culture. The cells secreted albumin, transferrin and -fetoprotein consistently for more than 100 days, to a maximum of 150 days. Thus, we have established a long-term culture of hepatocytes from human adults, which will be useful for basic studies of liver physiology such as metabolism and morphogenesis, as well as for other applications in the study of infectious hepatitis, pharmacology, pharmacokinetics, and toxicology.