Modulation of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithelial cell line

DEVELOPMENTAL

BIOLOGY

105, 211-220 (1984)

Modulation of Human Fetal Hepatocyte Survival and Differentiation by Interactions with a Rat Liver Epithelial Cell Line CHRISTIANE GUGUEN-GUILLOUZO,’ BRUNOCLEMENT,GERARDLESCOAT, DENISE GLAISE, AND ANDRE GUILLOUZO Unite de Recherche Hepatologique,

U 49, INSERM,

Hopital

Pontchaillou,

Received August 22, 198.7; accepted in revised

3.5011 Rennes Cedex, France

form May 4, 1984

Fetal human hepatocytes were isolated by collagenase digestion of liver fragments and cultured either alone or mixed with rat liver epithelial cells. Whereas they did not survive more than 2-3 weeks and showed rapid morphologic and functional alterations in conventional culture, fetal hepatocytes survived and retained or reverted to a globular morphology for several weeks and showed active albumin secretion for at least 13 days when cultured with rat liver cells. Increased levels of secreted albumin correlated with deposition of an insoluble extracellular material containing fibronectin and type III collagen located principally between the two cell types and around parenchymal cells. These observations show that fetal human hepatocytes are able to interact in vitro with another epithelial liver cell type obtained from a divergent species and that these cell-cell interactions influence both hepatocyte survival and expression of albumin. INTRODUCTION

Human liver differentiation during the fetal life remains poorly understood. Attempts to cultivate human fetal hepatocytes have shown that these cells like immature and mature mammalian ones (Bissell and Tilles, 1971; Leffert and Paul, 1972; Sell et aL, 1975; Acosta et aZ., 1978; Armato et aZ., 1978; Guguen-Guillouzo et aL, 1980a,b;Bissell, 1981) had a limited proliferative capacity and exhibited phenotypic alterations leading to decline or loss of the most specific functions within a few days. The cultures were made from either explants (Hillis and Bang, 1962; Noyes, 1973) or cells isolated using trypsin (Zuckerman et uL, 1967; Bissell and Tilles, 1971) or collagenase (Guguen-Guillouzo et uL, 1980a). However, it has been shown that specific cellular interactions are required for normal morphogenesis of various organs. In the liver, hepatocytes do not differentiate from the endoderm unless contact is established with the mesoderm which can be of nonhepatic origin or from divergent species (Le Douarin, 1975). Recently, we demonstrated that adult hepatocytes had an extended cell viability and expressed active differentiated functions for several weeks when cocultured with another epithelial cell type derived from rat liver (Guguen-Guillouzo et al, 1983b), suggesting that epithelial-epithelial interactions could occur in the liver. Indeed, although their precise origin remains questioned, these rat liver epithelial cells (RLEC) presumedly derive from terminal biliary ductular cells (Grisham, 1980; Guguen-Guillouzo and Guillouzo, 1983). ’ To whom correspondence

should be addressed. 211

Since interactions between hepatocytes and another liver epithelial cell have never been reported during embryonic development, the question arose as to whether liver epithelial cells could influence in vitro functional activities of human fetal hepatocytes and whether such influences were species specific. Although easy to isolate from human liver tissue, cell lines are usually composed of nonepithelial cells (Demoise et uL, 1971; Kaighn and Prince, 1971; Guillouzo et al, 1972; Tsiquaye et uk, 1978; Schaeffer and Kessler, 1980). The epithelial cell line obtained by Nakagomi and Ishida (1980) from a fetal human liver, was found to undergo rapid neoplastic transformation. Therefore, in this study, human fetal hepatocytes were associated only with RLEC. We found that these two cell types could interact together which led to an extended parenchymal cell survival and a stimulated albumin secretion rate if the cultures were maintained in presence of corticosteroids. MATERIAL

AND

METHODS

Cell isolation and culture. Hepatocytes were isolated from the liver of three fetuses aged 15,15, and 22 weeks, respectively, according to the procedure previously described (Guguen-Guillouzo et ah, 1980a). Briefly, after three washes with Hepes (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) buffer, liver fragments were digested with a Hepes-buffered solution containing 0.025% collagenase (Worthington CLS) and 5 mM CaC12, pH 7.6, under gentle stirring at 37°C. Three incubations of 10 min each with the enzymatic solution were performed. 0012-1606/84 $3.00 Copyright All rights

0 1984 by Academic Press, Inc. of reprodurtion in any form reserved.

212

DEVELOPMENTAL BIOLOG:Y

Hepatocyte suspensions were seeded at the density of 3 X lo6 cells in 4 ml of arginine-free medium supplemented with 10% fetal calf serum in 25cm2 polystyrene flasks. For cocultures, RLEC were added 4 hr or 3 or 7 days after hepatocyte seeding at the density of 2 X lo6 cells per 25cm2 flask. RLEC do not express hepatocytic functions and were not transformed; they derived from lo-day-old Fisher rat livers (Morel-Chany et ah, 1978). The medium was renewed 4 hr after hepatocyte seeding and every day thereafter. At this time, it was supplemented with hydrocortisone hemisuccinate (Roussel, France); its concentration was 7 X 10m5and 3.5 X lop6 M in pure and mixed cultures, respectively. The concentrations of hydrocortisone used were those which gave maximum cell viability and functional activities. Morphological studies. Living cultures were examined daily under phase-contrast microscopy. For electron microscopy studies, cultured fetal hepatocytes were fixed in 2.5% glutaraldehyde buffered with 0.1 Msodium cacodylate for 5 min, postfixed in a 1% osmium tetroxide solution, dehydrated, and embedded in Epon. Albumin assay. The amount of albumin secreted into the medium was determined by immunonephelometry (Ritchie, 1975). Standard human albumin solutions and culture media were mixed with appropriate dilutions of anti-albumin antibodies in 0.1 M phosphate buffer containing 2% polyethylene glycol and incubated at room temperature for 1 hr before measuring light scatter. Standard human albumin and anti-albumin antibodies prepared for immunonephelometry use were from

VOLUME 105,1984

ATAB, Scarborough, Maine. The reliability of immunonephelometry for the assay of albumin in the culture medium was confirmed by the following criteria: (1) absence of detectable cross reactivity with other proteins present in the medium, including rat and bovine albumin; (2) absence of detectable albumin in the medium of pure RLEC maintained in the same culture conditions; (3) addition to the medium of increasing amount of human albumin ranging from 2 to 45 pg/ml resulted in identical increases by immunonephelometry. The lower limit of sensitivity of the assay was 0.5-l pg/ml. Retie&in staining. The cells were fixed in situ in a mixture of 4% paraformaldehyde-0.2% glutaraldehyde buffered with 0.1 M sodium cacodylate for 15 min at 4°C (Guguen-Guillouzo et ah, 1983b) and reticular fibers were visualized using the silver impregnation medium (Gordon and Sweets, 1936). Fibronectin and collagen immunolocalixation. After in situ fixation with a 4% paraformaldehyde solution buffered with 0.1 M sodium cacodylate for 45 min at 4”C, cultures were incubated with specific antibodies against fibronectin, type I or type III collagens, provided by Dr. J. A. Grimaud (Lyon). As a second antibody, fluoresceinlabeled immunoglobulins were used. Controls consisted of incubated hepatocyte cultures with normal y-globulins and then with fluorescein-labeled anti-immunoglobin antibodies. RESULTS

The three fetal livers gave similar results. More than 90% of fetal human hepatocytes isolated by collagenase

FIG. 1. Morphology of fetal human hepatocytes (H) in primary culture. Parenchymal cells were maintained alone (A) or mixed with RLEC (B, C). Day 6 (A, B) or 12 (C) of culture. RLEC were seeded 4 hr after parenchymal cells. After some days of coculture, fetal hepatocytes tend to exhibit a more globular shape (C). (A-C) X240. All figures were obtained from the two 15-week-old fetuses.

GUGUEN-GUILLOUZOET AL.

Liver

Cell Interactions

in Culture

213

FIG. 2. Electron microscopic appearance of fetal human hepatocytes cultured in conventional conditions. On Day ‘7 of culture, the cells still contained flattened cisternae of rough endoplasmic reticulum (RER) and glycogen particles (gl) (A). Intracellular bundles of microfilaments (mf) are particularly abundant beneath the plasma membrane facing the medium (B). (N, nucleus; M, mitochondrion; d, desmosomal complexes.) (A) X18,500; (B) X12,000.

214

DEVELOPMENTALBIOLOGY

VOLUME105,1984

GUGUEN-GUILLOUZO ET AL.

Liver

dissociation were viable as indicated by the trypan blue exclusion test and their refringence under phase-contrast microscopy. Within 3 hr, 60 to 80% of these cells attached to plastic, reaggregated, and then formed monolayers of spread granular epithelial cells (Fig. 1A). In conventional conditions, they underwent a few divisions and began to detach around the end of the first week. Cell survival did not exceed 2-3 weeks (GuguenGuillouzo et aZ.,1980a). In coculture, RLEC actively divided and formed a confluent monolayer with hepatocytes within 24-48 hr after their addition (Fig. 1B). Once cell confluency was reached, no obvious change was observed in the cocultures during 4-6 weeks. Human fetal hepatocytes retained or progressively reverted to a more globular morphology whereas RLEC appeared as spread as in the absence of parenchymal cells. After a few days of coculture, fetal hepatocytes formed groups of packed cells (Fig. 1C). When put in conventional culture, fetal hepatocytes retained their typical fine structure for 4-6 days. Later on, they appeared loaded with fibrils either randomly distributed in bundles into the cytoplasm or forming a layer beneath the plasma membrane not in contact with another cell or the substratum (Figs. 2A and B). Some well-developed Golgi complexes were located in the vicinity of bile canaliculi. The presence of fibrillar extracellular material surrounding the hepatocytes was very occasional. In contrast, cocultured fetal human hepatocytes did not exhibit obvious ultrastructural changes during the first 2-3 weeks. The rough endoplasmic reticulum was abundant and surrounded mitochondria; glycogen particles were numerous and some Golgi complexes were present near bile canaliculi. (Figs. 3A and 4). As early as 24 hr after RLEC addition, discontinuous close contacts were established between the two cell populations and by Days 4 to 6, deposits of extracellular material were observed in intercellular spaces. This material was formed of randomly oriented fibrillar components and in places of a discontinuous amorphous layer (Figs. 5A and B). As the cultures aged, the amount of fibrillar material increased while intimate contacts between hepatocytes and RLEC tended to disappear (Fig. 5C). Intracytoplasmic fibrils remained limited in number even beneath the plasma membrane and after 3 weeks of culture (Figs. 3B and 5C). Some lysosomes and lipid droplets were observed. RLEC exhibited a fine structure very different from that of hepatocytes. They contained

Cell Interactions

in Culture

FIG. 4. Electron microscopic appearance of fetal human hepatocytes cocultured with RLEC. Note the presence of a Golgi apparatus (GA) in the vicinity of a bile canaliculus (BC), (tj, tight-junction; d, desmosome); Day 7 of coculture. X13,500.

fewer organelles, smaller mitochondria, and short and often dilated cisternae of rough endoplasmic reticulum. Glycogen particles were never observed (Fig. 5A). The extracellular material deposited was analyzed in both mixed and pure cultures by the silver impregnation method and immunofluorescence. In coculture, reticulin fibers were seen principally in hepatocyte islands (not shown). Fibronectin, already abundant on Day 3, displayed fibers from hepatocyte cords (Fig. 6A). A few deposits of type III collagen were visualized, mainly between the two cell populations and in hepatocyte cords (Fig. 6B). Type I collagen was rare or absent. In pure cultures of hepatocytes, reticulin fibers were virtually absent while in those of RLEC, they were scarce, at least during the first 10 days of cell confluency. Only fibronectin was detected in pure hepatocyte cultures.

FIG. 3. Electron microscopic appearance of fetal human hepatocytes cocultured with RLEC. (A) Day 7 of coculture. Hepatocytes contain numerous organelles; glycogen particles (gl) are abundant. Microfilaments (mf) are scarce. (N, nucleus; M, mitochondrion; GA, Golgi apparatus.) X 11,000. (B) Day 24 of coculture. The fine structure of the hepatocytes is still well preserved. Note the absence of accumulation of microfilaments even beneath the plasma membrane (arrow) which displays microvilli (v) into the nutrient medium. These hepatocytes likely correspond to a group of packed rounded cells (see Fig. 1C). X11,000.

216

GLIGUEN-GUILLOUZO ET AL.

Liver

Cell Interactions

in Culture

FIG. 6. Immunofluorescence localization of fibronectin and type III collagen in cocultures of fetal human hepatocytes. (A) Fibronectin is mainly located in hepatocyte islands but formed fibers which extend in RLEC areas. (B) Type III collagen is virtually present only in hepatocyte cords. (A) X140; (B) X175.

The albumin secretion rate dramatically declined on Day 6 in conventional culture while it rapidly increased in coculture reaching by Day 8 a maximum equal to three times its initial value (Fig. 7). Moreover, similar increased albumin levels were obtained when RLEC were added to pure hepatocyte cultures which showed decreased albumin production, i.e., on Days 3 or 7 after hepatocyte seeding. By Day 13 of culture, the albumin secretion rate declined whatever the time of addition of RLEC (Fig. 7). DISCUSSION

Our data demonstrate that when cocultured with RLEC, fetal human hepatocytes survived longer and secreted albumin, a specific liver plasma protein, at much higher amounts than in pure cultures which showed rapid morphologic and functional alterations, as previously reported (Bissell and Tilles, 1971; Guguen-Guillouzo et al., 1980a). The differences between pure and mixed cultures cannot be entirely explained by a different proliferative rate of the hepatocytes. Active albumin production was similarly reexpressed in 7 days cultured hepatocytes. The capability of hepatocytes to revert in vitro to a higher differentiated state indicates that in coculture, these cells retained their biosynthetic activity. Therefore, it can be assumed that hepatocytes undergo in vitro modifications in the control of the cellular biosynthetic machinery rather

than irreversible structural alterations and that under an appropriate environment, specific functions may be reincreased in cultured fetal hepatocytes. Similar findings were made by Houssaint (1976) with primary cultures of 8-day quail embryo hepatocytes. She reported a shift from fibroblastic to initial epithelial shape with intracellular accumulation of glycogen after late addition of hepatic or pulmonary mesenchyme. Since interactions between mesenchyme and epithelium are required for normal morphogenesis of various organs including liver (Le Douarin, 1975), it could be questioned as to whether RLEC have a mesenchymal origin. Recently, Grisham (1980) reviewed their characteristics and concluded that they most likely derived from terminal biliary ductular cells rather than from endothelium (Sirica and Pitot, 1980). It must be pointed out that epithelial-epithelial interactions have already been described (Hay, 1981). Our observations give further evidence that RLEChepatocyte interactions are not species specific. The means by which such interactions occur, remains to be elucidated. One can envision that cell contact is required between the two cell types and that a stimulus is then propagated between adjacent cells. Alternatively, a diffusible molecule, perhaps a component of the extracellular material, could mediate such interactions. The cellular source of the insoluble extracellular material remains unclear. Both hepatocytes (Voss et al, 1979; Diegelmann et ak, 1980; Tseng et al, 1982) and RLEC

FIG. 5. Electron microscopic appearance of extracellular material deposited in cocultures of fetal human hepatocytes and RLEC. (A and B) Day 7 of coculture. Intimate contacts (c) are visible between RLEC and hepatocytes (H). The extracellular material is formed of fibrillar material (FM) randomly distributed between the two cell types. Note the presence of dilated rough endoplasmic reticulum (RER) and smaller mitochondria (m) in RLEC (A). In places (B), the insoluble material forms a layer (arrow) between the two cell types. (A) X12,000; (B) X20,000. (C) Day 24 of coculture. Increased amount of fibrillar material (FM) is observed between an hepatocyte (H) and a RLEC. (gl, glycogen particles; mf, microfilaments; 1, lipid droplet.) ~20,000.

218

DEVELOPMENTAL BIOLOGY

VOLUME 105. 1984

soluble matrix. Dexamethasone has been reported to increase fibronectin synthesis by fetal rat hepatocytes in primary culture (Marceau et al, 1980). Hydrocortisone could similarly increase accumulation of this glycoprotein in cocultures of fetal human hepatocytes. Cell shape is greatly affected by the nature of the support. Whereas they retain for several days their cuboidal morphology on collagen gels (Sirica et aL, 1979), hepatocytes become rapidly flattened on plastic, collagen (Michalopoulos and Pitot, 1975), fibronectin (Marceau et aL, 1982; Guguen-Guillouzo et aL, 1982), confluent lung fibroblasts (Michalopoulos et aL, 1979), liver biomatrix (Rojkind et aL, 1980), or the extracellular material sei2 1'4 1'6 k t k io ;t i creted by confluent cultures of cornea1 bovine endothelial t cells (Guguen-Guillouzo et aL, 1982). In the last study, Days of culture an inverse relationship was found between the degree FIG. ‘7. Albumin secretion by fetal human hepatocytes cultured alone of cell spreading and the maintenance of hepatic funcor in association with RLEC. Arrows indicate time of addition of tions. Modifications of cell shape involve alterations of RLEC: 4 hr, 3 or 7 days. Albumin secretion into the medium was the cytoskeleton. measured daily in pure hepatocyte cultures (0) and in cultures mixed with RLEC 4 hr (o), 3 days (m), or 7 days (A) after hepatocyte seeding. In parallel with loss of specific functions, accumulation The values are expressed in pg/ml of medium. Each point represents of intracellular fibrils, particularly beneath the plasma the mean of triplicate cultures. The differences did not exceed 10%. membrane facing the culture medium, rapidly occurs in hepatocyte cultures (Chapman et aL, 1973; Guguen et aL, 1975; Mak et aL, 1980). When in coculture, fetal he(Sakakibara et a& 19’78;Foidart et aL, 1980) have been patocytes retained a globular morphology (more than shown to produce collagen and fibronectin when main- adult cocultured cells) and accumulated much fewer fitained in pure culture. However, no insoluble material brils. Moreover, Golgi complexes which were hypertrostained by the silver impregnation method was observed phied, as this is the case in in viva fetal hepatocytes, in pure cultures. This is consistent with the findings were often located near bile canaliculi. These findings that collagen newly synthesized by cultured hepatocytes favor the conclusion that in order to express a differis rapidly degraded (Diegelmann et a!, 1980) and that entiated state, hepatocytes must remain cuboidal and untransformed RLEC do not accumulate insoluble ex- polarized. tracellular material until they have been left in a conIn conclusion, these results extend to fetal hepatocytes fluent sheet for several days (Sakakibara et aL, 1978; our previous observations with cultured adult hepatoKarasaki and Raymond, 1981). In coculture, hepatocytes cytes emphasizing the role played by specific cell-cell probably play a key role in the deposition of the extra- interactions in long term in vitro expression of specific cellular material. Indeed, the material deposited in co- liver functions. This coculture model would be presumcultures of fetal human hepatocytes differs, both in its ably suitable for better understanding liver differendistribution and in the relative amounts of immunolo- tiation during embryonic life, which is associated with calized components from that found in cocultures of imbalance between fetal proteins and their adult counadult human (Guguen-Guillouzo et aL, 1983a) and rat terparts. (Guguen-Guillouzo et al, 1983b) hepatocytes. We are indebted to Dr. J. Mention and Dr. M. F. Dubois for providing Interactions of RLEC with fetal human hepatocytes with liver samples, Dr. J. A. Grimaud for the gift of anti-type I as well as with adult hepatocytes (Baffet et aL, 1982) us and anti-type III collagen and fibronectin antibodies, Mrs. M. Rissel are corticosteroid dependent. These hormones are known for preparing the illustrations, and Mrs. A. Vannier for typing the to have a variety of beneficial effects on both viability manuscript. This work was supported by the Institut National de la and functional activities of liver cells in culture (Lam- Sante et de la Recherche MBdicale and the FBdLration Nationale des biotte et aL, 1973; Guguen et aL, 1974, 1975; Murison, Centres de Lutte contre le Cancer. 1975; Laishes and Williams, 1976; Williams et CAL,1978; REFERENCES Guguen-Guillouzo et aL, 1980b; Marceau et aL, 1980). However, the molecular basis of their effects remains ACOSTA, D., ANUFORO, D. C., and SMITH, R. V. (19’78). Primary monolayer cultures of postnatal rat liver cells with extended differentiated poorly understood. Corticosteroids could act at the functions. In Vitro 14, 428-435. plasma membrane level by inducing establishment of ARMATO, U. P. G., ANDREIS, E., DRAGHI, E., NEGRI, L., MENGATO,L., intimate contacts between the two cell types and by and NERI, G. (1978). Studies on the persistence of differentiated functions in rat hepatocytes set into primary tissue culture. II. stimulating production of certain elements of the in-

GUGUEN-GUILLOUZOET AL.

Liver

Production of specific exportable proteins and the effect of purine cyclic nucleotides: An immunofluorescence study. In Vitro 14, 838847. BAFFET, G., CLEMENT,B., GLAISE, D., GUILLOUZO,A., and GUGUENGUILLOUZO,C. (1982). Hydrocortisone modulates the production of extracellular material and albumin in long-term co-cultures of adult rat hepatocytes with other liver epithelial cells. &o&em Biophys. Res. Commun 109, 507-522. BISSELL, D. M. (1981). Primary hepatocyte culture: Substratum requirements and production of matrix components. Fed Proc. 40, 2469-2473.

BISSELL,D. M., and TILLES, J. G. (1971). Morphology and function of cells of human embryonic liver in monolayer culture. J. Cell Biol 50,222-231. CHAPMAN,G. S., JONES,A. L., MEYER,U. A., and BISSELL,D. M. (1973). Parenchymal cells from adult rat liver in non proliferating monolayer culture. II. Ultrastructural studies. J. Cell Biol 59, 735-747. DEMOISE,C. F., GALAMBOS,J. T., and FALEK, A. (1971). Tissue culture of adult human liver. Gastroenterology 60, 390-399. DIEGELMANN,R. F., COHEN,I. K., and GUZELIAN,P. S. (1980). Rapid degradation of newly synthesized collagen by primary cultures of adult rat hepatocytes. Biochem Biophys. Res. Commun 97.819-826. FOIDART,J. M., BERMAN,J. J., PAGLIA, L., RENNARD,S., ABE, S., PERANTONI,A., and MARTIN, G. R. (1980). Synthesis of fibronectin, laminin, and several collagens by a liver-derived epithelial line. Lab.

Cell Interactimzs in Culture

219

HAY, E. (1981). Collagen and embryonic development. In “Cell Biology of Extracellular Matrix” (E. Hay, ed.), pp. 379-409. Plenum, New York. HILLIS, W. D., and BANG, F. B. (1962). The cultivation of human embryonic liver cells. Exp. Cell Res. 26, 9-36. HOUSSAINT,E. (1976). Reversibilitb de la diffhrenciation observbe dans les cultures cellulaires de foie embryonnaire d’oiseau. J. EmlnyoL Exp. MwphoL

36,227-240.

KAIGHN, M. E., and PRINCE,A. M. (1971). Production of albumin and other serum proteins by clonal cultures of normal human liver. Proc. NatL Acad Sci USA 68, 2396-2400.

KARASAKI, S., and RAYMOND,J. (1981). Formation of intercellular collagen matrix by cultured rat liver epithelial cells and loss of its ability in hepatocarcinogenesis in vitro. D@rentiation 19, 21-30. LAISHES,B. A., and WILLIAMS, G. M. (1976). Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12, 821-831.

LAMBIOTTE,M., VORBRODT, A., and BENEDETTI,E. L. (1973). Expression of differentiation of rat foetal hepatocytes in cellular culture under the action of glucocorticoids. Appearance of bile canaliculi. Cell mer. 2, 43-53. LE DOUARIN,N. M. (1975). An experimental analysis of liver development. Med BioL 53,427-455. LEFFERT,H. L., and PAUL, D. (1972). Studies on primary cultures of differentiated fetal liver cells. J. Cell BioL 52, 559-568. Invest. 42,525-532. GORDON,H., and SWEETS,H. H., JR. (1936). A simple method for the MAK, W. W., SATTLER,C. A., and PITOT, H. C. (1980). Accumulation of actin microfilaments in adult rat hepatocytes cultured on collagen silver impregnation of reticulum. Amer. J. PathoL 12,545-551. GRISHAM,J. W. (1980). Cell types in long term propagable cultures gel/nylon mesh. Cancer Res. 40, 4552-4564. of rat liver. Ann N. Y. Acad Sci 349, 128-137. MARCEAU,N., GOYEITE, R., VALET, J. P., and DESCHENES,J. (1980). GUGUEN,C., GUILLOUZO,A., LENOIR,P., and BOUREL,M. (1974). Action The effect of dexamethasone on formation of a fibronectin extraof hydrocortisone hemisuccinate on newborn rat liver explant culcellular matrix by rat hepatocytes in vitro. Exp. Cell Res. 125,497tures. Biomedicine 21, 286-292. 502. GUGUEN,C., GUILLOUZO,A., BOISNARD,M., LE CAM, A., and BOUREL, MARCEAU,N., NOEL, M., and DESCHENES,J. (1982). Growth and funcM. (1975). Etude ultrastructurale de monocouches d’hbpatocytes de tional activities of neonatal and adult rat hepatocytes cultured on rat adulte cultiv6s en prisence d’hkmisuccinate d’hydrocortisone. fibronectin-coated substratum in serum-free medium. In Vitro 18, BioL GastroenteroL 8, 223-231. l-11. GUGUEN-GUILLOUZO,C., and GUILLOUZO,A. (1983). Modulation of MICHALOPOULOS, G., and PITOT, H. C. (1975). Primary culture of pafunctional activities in cultured rat hepatocytes. MoL Cell B&hem. renchymal cells on collagen membranes: morphological and bio53/54, 35-56. chemical observations. Exp. Cell Res. 94, 70-78. GUGUEN-GUILLOUZO, C., BAFFET,G., CLEMENT,B., BEGUE,J. M., GLAISE, MICHALOPOLILOS, G., RUSSEL,G., and BILES,C. (1979). Primary cultures D., and GUILLOUZO,A. (1983a). Human adult hepatocytes: Isolation of hepatocytes on human fibroblasts. In Vitro 15, 796-806. and maintenance at high levels of specific functions in a co-culture MOREL-CHANY,E., GUILLOUZO,C., TRINGAL, G., and SZAJNERT,M. F. system. In “Isolation, Characterization and Use of Hepatocytes” (1978).“Spontaneous” neoplastic transformation in vitro of epithelial (R. A. Harris and N. W. Cornell, eds.), pp. 105-110. Elsevier, New cell strains of rat liver: cytology, growth and enzymatic activities, York. Eur. J. Cancer 14, 1341-1352. GUGUEN-GUILLOUZO, C., CLEMENT,B., BAFFET, G., BEAUMONT,C., Mo- MURISON,G. L. (1975). Growth of fetal rat liver cells in response to REL-CHANY,E., GLAISE,D., and GUILLOUZO,A. (1983b). Maintenance hydrocortisone. Exp. Cell Res. 100, 439-443. and reversibility of active albumin secretion by adult rat hepatocytes NAKAGOMI,O., and ISHIDA, N. (1980). Establishment of a line from a co-cultured with another liver epithelial cell type. ,%p. Cell Ra. human fetal liver and its xenotransplantation to nude mice. Gann 143,47-54. 71,213-219. GUGUEN-GUILLOUZO, C., MARIE, J., COTTREAU,D., PASDELOUP,N., and NOYES,W. F. (1973). Culture of human fetal liver. Proc. Exp. BioL KAHN, A. (1980a). Isosyme shift in cultured fetal human hepatocytes: Med. 144,245-248. a study of pyruvate kinase and phosphofructokinase. B&hem. BioRITCHIE, R. F. (1975). Automated immunoprecipitation analysis of phys. Res. Commun. 93.528-534. serum proteins. In “The Plasma Proteins: Structure, Function and GUGUEN-GUILLOUZO, C., SEIGNOUX,D., COURTOIS,Y., BRISSOT,P., MARGenetic Control” (F. W. Putnam, ed.), Vol. II, pp. 375-425. Academic CEAU,N., GLAISE,D., and GUILLOUZO,A. (1982).Amplified phenotypic Press, New York. changes in adult rat and baboon hepatocytes cultured on a complex ROJKIND,M., MACKENSEN,S., GIAMBRONE,M. A., PONCE,P., and REID, biomatrix. BioL Cell 46, 11-20. L. M. (1980).Connective tissue biomatrix: Its isolation and utilization GUGUEN-GUILLOUZO, C., TICHONICKY,L., SZAJNERT,M. F., and KRUH, for long-term cultures of normal rat hepatocytes. J. Cell BioL 87, J. (1980b). Changes in some chromatin and cytoplasmic enzymes of 255-283. perinatal rat hepatocytes during culture. In Vitro 16, l-10. SAKAKIBARA,K., TAKAOKA,T., KATSUTA,H., UMEDA,M., and TSUKADA, GUILLOUZO,A., GUGUEN,C., LENOIR,P., and BOUREL,M. (1972). ModY. (1978).Collagen fiber formation as a common property of epithelial ifications morphologiques et ultrastructurales l&s i la sbnescence liver cell lines in culture. Exp. Cell Res. 111, 63-71. dans les lignkes de foie humain adulte. C, R. Acad Sci. (Par&) 274, SCHAEFFER,W. I., and KESSLER,D. J. (1980). Human liver cells in 484-487. culture. Methods Cell BioL 21 B, 457-473.

220

DEVELOPMENTAL BIOLOGY

SELL, S., SKELLY, H., LEFFERT, H. L., MULLER-EBERHARD, U., and KIDA, S. (1975). Relationship of the biosyntheses of cy-fetoprotein, albumin, hemopexin, and haptoglobin to the growth stage of fetal rat hepatocyte cultures. Ann N. Y Acad Sci 259, 45-58. &RICA, A. E., and PITOT, H. C. (1980). Drug metabolism and effects of carcinogens in cultured hepatic cells. PharmucoL Re-u. 31, 205228. &RICA, A. E., RICHARDS, W., and TSUKADA, Y. (1979). Fetal phenotypic expression of adult rat hepatocytes on collagen gel/nylon meshes. Proc. Natl. Acad. Sti USA 76, 283-287. TSENG, S. C. G., LEE, P. C., ELLS, P. F., BISSELL, D. M., SMUCKLER, E. A., and STERN, R. (1982). Collagen production by rat hepatocytes and sinusoidal cells in primary monolayer culture. Hepatolcgy 2, 13-18.

VOLUME 105, 1984

TSIQUAYE, K. N., DAS, P. K., and ZUCKERMAN, A. J. (1978). Long term cultivation of active human foetal liver cells. Brit. J. Exp. Pathol 59,482-488. Voss, B., ALLAM, S., RAUTERBERG, J., ULLRICH, K., GIESELMANN, V., and VON FIGURA, K. (1979). Primary cultures of rat hepatocytes synthesize fibronectin. B&hem. Biophys. Res. Commun SO, 13481354. WILLIAMS, G. M., BERMUDEZ, E., SAN, R. H. C., GOLDBLATT, P. J., and LASPIA, M. F. (1978). Rat hepatocyte primary cultures: IV. Maintenance in defined medium and the role of production of plasminogen activator and other proteases. In Vitro 14, 838-848. ZUCKERMAN, A. J., TSIQUAYE, K. N., and FULTON, E. (1967). Tissue culture of human embryo liver cells and the cytotoxicity of aflatoxin B,. Brit. J Exp. Pathol. 48, 20-27.