MXPA98007554A - Cellular cultivation media for mamif cells - Google Patents

Abstract

A chemically defined mammalian cell culture medium is provided that supports the long-term maintenance and clonal development of mammalian hepatocytes and other cells.

Description

CELLULAR CULTIVATION MEDIA FOR MAMMALIAN CELLS FIELD OF THE INVENTION The present invention relates generally to cell culture media for mammalian cells. In particular, the invention relates to cell culture media that allow the expansion and long-term maintenance of a cellular population of mammalian hepatocytes, cell lines derived from hepatocytes, malignant lines derived from hepatocytes, and other cells.

BACKGROUND OF THE INVENTION It is well known that specific cell lines can be developed in vitro in optimally formulated nutrient or culture media. Examples of some culture media developed for special purposes are: RPMI 1640 medium for the development of human B lymphoid cells and malignant cells, Changs medium for the development of amniotic fluid cells, Medium 199 for the development of fibroblast cells of Mouse, Minimum Essential Medium (MEM) a REF. 28427"minimal" medium for the development of bound mammalian cells, and Leibovitz medium for development in the absence of C02. Such diverse media are distinguished from each other in that they contain critically different components in precise amino acids, vitamins, organic salts, trace elements and other organic compounds that promote the maximum development of cultured cells. For the development of mammalian cells the chemically defined media are usually supplemented with various sera, preferably fetal calf serum or neonatal calf serum, and other incompletely defined growth factors. A major drawback for the serum, however, is that its constituents can vary widely, thereby introducing undefined biological components into the nutrient medium, which contributes to the variability of biochemical and cellular events. In addition, the serum is expensive and can result in critical immune reactions in patients if the cells are used for clinical purposes. The present invention is mainly directed to the culture of mammalian hepatocyte cells, using chemically defined means that allow the long-term expansion of the cell population. The term "chemically defined media" is used in tissue culture to refer to culture media of known chemical composition, both quantitatively and qualitatively, in contrast to media containing natural products such as animal serum. Liver regeneration is achieved mainly by the cell division of mature adult hepatocytes, as reported by Grisham, J.W. et al., Cancer Res. 22_: 842 (1962) the description of which is incorporated by reference herein. These cells, or a fraction thereof, have a high capacity for clonal development, as shown by hepatocyte transplant experiments in "ectopic sites (Jirtle, RL et al., Cancer Res. 42: 3000 (1982), the description of which is incorporated by reference herein) and into transgenic mouse models (Rhim, JA et al., Science 263: 1149 (1994), the description of which is incorporated by reference herein). shown in various studies, however, that when the liver is stimulated to regenerate while the proliferation of mature hepatocytes is suppressed, facultative totipotential cells emerge and proliferate See, for example, Thorgeirsson, SS et al., Proc. Soc. Exp. Biol. Med. 204: 253 (1993), the description of which is incorporated by reference herein.Such cells, sometimes referred to as "oval cells" can mature into hepatocytes in animal models of finite or ductular structures composed of cells ("ductular hepatocytes") with mixed hepatocytes and epithelial markers of the bile duct. See, Gerber, M.A. and collaborators, Amer. J. Path. 110: 70 (1983) 'and Vanders teenhoven, A.M. and collaborators, Arch. Pathol. Lab. Med. 114: 403 (1991), the descriptions of which are incorporated by reference herein. Little is known, however, regarding its origin and with respect to the controls that regulate its phenotypic transitions to hepatocytes or ductular cells. Despite the high capacity of hepatocytes to proliferate in vi, the growth of totipotent cells directly or facultatively, the conditions that determine their growth potential and their phenotypic transitions are not completely understood, due solely to the limited success in the development of the hepatitos in primary culture. It is typically the case that hepatocytes in primary culture under the influence of primary mitogens enter one or two divisions, and then the cells degenerate and die. Until now, several researchers have failed to develop a medium that allows hepatocytes to proliferate and survive. For example, Berry, N.M. and collaborators, J ^ Cell. Biol. 43_: 506 (1969), the description of which is incorporated by reference herein, showed the collagenase perfusion technique that allows the liver tissue to dissociate into its component cellular elements, based on size. Subsequently, Bissell, D.M. and collaborators, J. Cell. Biol. 59 (3): 722 (1973) and Bonney, R.J. and collaborators, In Vitro 9_: 39"9 (1974), the descriptions of which are incorporated by reference herein, described the first methods for the culture of isolated hepatocytes which may have survived for one or two days. Long-term hepatocytes on collagen gels for a maximum of 7 to 10 days was reported by Michalopoulos, G. et al., Exp. Cell. Res. 94 (1): 70 (1975), the description of which is incorporated by reference herein The common feature of all the systems referred to above is that the hepatocytes in those systems were kept in culture without any evidence of cell proliferation. maintenance of non-proliferating cells for a brief period The first successful attempt to initiate DNA synthesis in hepatocytes used the newly discovered epidermal growth factor (EGF) to then, as reported by Richman, R.A. and collaborators, Proc. Nat. Acad. Sci. USA 73: 3589 (1976), the description of which is incorporated by reference herein. In subsequent years other diverse groups of researchers used EGF as a mitogen for hepatocytes and reported regarding the mitogenic effects of EGF and its modulation by other factors such as, for example, matrices such as Type I collagen, zinc, and proline. The growth factor of hepatocytes, also known as dispersion factor (hereinafter referred to as "HGF" or "HGF / SF") was discovered, cloned and finally sequenced in the late 1980s. See Michalopoulos, G. and collaborators, Federation Proceedings 42: 1023 (1983); Michalopoulos, G. et al., AACR Proceedlngs 2_4_: 58 (1983); Michalopoulos, G. et al., Cancer Res. 44 (10): 4414 (1984); and Miyazawa, K. and collaborators, Biochem. Bj.ophys. Res. Common. 163: 967 (1989) the descriptions of which are incorporated by reference herein. It was found that HGF / SF is a mitogen for many hepatocytes, as well as for epithelial cells. The importance of HGF / SFs for the liver is due to the fact that this is the trigger for the regeneration of the liver through an endocrine mechanism. Recently, several studies have shown that HGF / SF, the epidermal growth factor ("EGF") and the transforming growth factor a ("TGFa") are the primary mitogens for hepatocytes in culture, by the stimulation of limited DNA synthesis in hepatocytes in chemically defined media. See, for example, Michalopoulos, G.K., Fed. Am. Soc. Exp. Biochem J. 4_: 17ß (1990), the description of which is incorporated by reference herein. It was subsequently found that these growth factors additionally play a role in the regeneration of the liver after partial hepatectomy. Injection of HGF / SF, TGFa, or EGF in rats induces DNA synthesis in hepatocytes. See, for example, Liu, M.L. and collaborators, Hepatology 1_9: 1521 (1994), the description of which is incorporated by reference herein. In all these systems, however, it was reported that hepatocytes entered DNA synthesis and mitosis only for a limited time, typically 1 to 3 days. After 1 to 2 rounds of DNA synthesis and cell division, the cultures were degenerated and all cells died in approximately 7 to 10 days. Up to now, there has been no documented expansion of the number of hepatocytes in cell culture by the addition of either EFG or HGF alone, or in combination. Cell replication in cultures that contain these growth factors is rather self-limited and the number of hepatocytes that die exceeds the number of hepatocytes that are newly generated. Cell replication in cultures containing other hepatocyte mitogens such as transforming growth factors, such as TGFα, and fibroblast acid growth factor is similarly self-limited.

More recently, Mita a, T. et al., Hepatology 13 (1): 21 (1991); Mitaka, T. et al., Hepatology 16 (2): 440 (1992); Mitaka, T. et al., Virchows Arch. B Cell Pathol. Incl. Mol. Pathol _62_: 329 (1992); - and Mitaka, T. et al., Cancer Res. 53..3145 (1993), the descriptions of which are incorporated by reference herein, have reported that the addition of nicotinamide, dexamethasone and EGF to a conventional culture medium resulted in the formation of colonies of small hepatocytes that arise in a dense culture of standard size parenchymal hepatocytes. In an additional study, Mitaka, T. et al., J. Cell. Physiol 157: 461 (1993), the description of which is incorporated by reference herein, have reported that the numbers of colonies induced by the combinations of EGF + HGF, EGF + TGF-a, and HGF + TGF-a not they were different from those of the colonies induced by each mitogen alone. In these studies, however, there was no significant expansion of the total cell population, nor evidence of clonal development, and there was loss of differentiation.

Until now, there has been no chemically defined medium, supplemented or not, that is capable of supporting the proliferation, differentiation and long-term viability of hepatocytes. While for many purposes the use of an undefined supplement is satisfactory, in cases where studies of the development, metabolism and / or differentiation of the cells in culture are carried out, it is more desirable to have a supplement that is defined. The introduction of indefinite components to a cell culture can contribute to the variability, inability to predict and contamination in the results of the study and applications of cell cultures. The use of defined means is particularly important and advantageous in areas of drug metabolism, artificial organ development, cell transplantation, gene therapy, and basic cell studies for research. The previously described limited capacity of hepatocytes to proliferate in primary culture has impeded long-term studies or uses that required long-term viability or proliferation. The applications of hepatocyte cultures to cell transplantation and gene therapy have been impeded in this way. Consequently, there remains a need for a chemically defined medium that will allow hepatocytes to proliferate and survive in the long term. Among the potential applications for such media and hepatocytes and other cells cultured as such, are gene therapy, bioartificial organs, cell transplants, drug production, and drug and chemical product testing. As stated above, the current state of the art does not provide a culture system for hepatocytes in which the hepatocytes expand as a cell population by sustained proliferation, and there is a need for such a system. The present invention provides a completely defined culture medium that allows sustained proliferation and long-term expansion of hepatocytes.

BRIEF DESCRIPTION OF THE INVENTION According to the present invention, a new chemically defined cell culture medium is provided. This medium supports the sustained clonal development of the primary hepatocytes and cell lines of hepatocytes, genetically transformed hepatocytes and hepatocytes obtained from neoplastic sources, resulting in the expansion of the cell population. This means also allows the complete differentiation of the metabolic, structural and secretory functions of the cells developed in it. Under these conditions, hepatocytes undergo multiple proliferative cycles. Once the confluence is reached, or in the presence of specific matrix components, nutrients and / or developmental factors, these proliferating cells stop dividing and maintain a mature hepatocyte phenotype for many months or more. Accordingly, a principal objective of the present invention is to provide a culture medium for the proliferation and sustained viability of the hepatocytes. Yet another objective of the present invention is to provide a culture medium for the differentiation and sustained viability of hepatocytes. Yet another objective of the present invention is to provide a culture medium for the sustained proliferation of hepatocytes, which are reversed to complete differentiation as development ceases. Yet another objective of the present invention is to provide a culture medium for the long-term expansion of hepatocytes, which does not contain serum, such that the medium is completely defined. Yet another objective of the present invention is to provide a culture medium for the proliferation, differentiation and sustained viability of hepatocytes on a variety of matrix substrates. These and other objects of the present invention are achieved by one or more of the following embodiments. In one aspect, the invention features a chemically defined HBM culture medium for the maintenance, differentiation and long-term development of mammalian hepatocytes, comprising: a) a synthetic reserve basal medium designed for the culture of mammalian cells; and b) a developmental amount of the hepatocyte cells, of components selected from nicotinamide, amino acids, transferrin, hormones, dexamethasone, trace metals, and simple carbohydrate selected from the group consisting of D-glucose and D-galactose, and any combination thereof. In a preferred embodiment the invention features a culture medium HBM which also comprises buffer, antibiotics and albumin. In yet another aspect, the invention features a culture medium for mammalian cells comprising the composition of HGM as defined in Tables I and II, wherein the reserve basal medium of Table I comprises a mixed DMEM such that the final concentration of D-glucose is preferably about 2.0 g / 1 and the amount of D-galactose is preferably about 2.0 g / 1. Other features and advantages of the invention will be apparent from the following description of the preferred embodiment, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1C are graphs showing the results of studies in which rat hepatocytes were cultured in HBM medium as described herein, supplemented with growth factors, as indicated. All the points? represent the mean and standard error of at least three separate crops.

Figure IA shows the percent of labeled nuclei (BRdU) of the proliferating cell cultures at different times after the isolation of the hepatocytes. Cells were grown in HBM supplemented with HGF / SF and EGF.

Figure IB shows the incorporation of tritiated thymidine [3 H] -thymidine (disintegrations per minute) into the DNA in cultures at different times after isolation of the hepatocytes. The cells were exposed to HGF / SF in HBM (O); EGF in HBM (O); HGF / SF + EGF in HBM (?); and a control, HBM only (D).

Figure 1C shows the amount of DNA per plate at different days of the cells grown in HBM medium alone (control) («); HGF / SF in HBM (?); EGF in HBM (V); and HGF / SF + EGF in HBM (•).

Figure 2 is a graph showing DNA on day 15 per plate of rat hepatocytes developed in HBM with the indicated growth factors. The cells represented by the control (day t = 0) and day 15 were developed in HBM without any growth factor.

Figure 3 is a graph showing the amount of DNA synthesis per plate (μg / culture) of rat hepatocytes at day 15 in HBM supplemented with HGF / SF and EGF developed on different matrices. "CC" means coated collagen; "ECL" is a commercial matrix; "m COLL IV" means IV mouse collagen; "CC VITROGEN" means bovine skin collagen type I; "POLY D LYS" means poly D-lysine; "LAMININ" means mouse laminin; "h FIBRONECT" means human fibronectin; and "m COL I" means mouse collagen I.

Figures 4A-4G are photographs of rat hepatocytes cultured in HBM, as described herein under various conditions.

Figure 4A shows the hepatocytes in HBM medium with HGF / SF and EGF on day 1 after isolation, showing the non-proliferating, subconfluent, typical hepatocytes.

Figure 4B shows the hepatocytes on day 4 in HBM medium induced with HGF / SF showing the typical diffuse morphology.

Figure 4C shows the cultures of hepatocytes that have reached confluence on day 15, showing typical morphology.

Figures 4D and 4E are photomicrographs of choice of cells in Figures 4A and 4C, respectively.

Figures 4F and 4G are photomicrographs of stained hepatocytes that were transfected on day 3 with a replication-defective retrovirus, containing lac-Z and stained for the expression of β-galactosidase as described further below, and then photographed on the day 1 (Figure 4F) and on day 10 (Figure 4G) after transfection The cells were grown in HBM medium supplemented with HGF / SF and EGF as described below Figures 5A and 5B are photographs of spotted Northern showing the expression of specific genes at different days in culture of rat hepatocytes maintained in HBM in the presence of HGF / SF and EGF as described herein.GAPDH expression and intensity of 28S RNA after staining with ethidium bromide, were used as internal controls.

Figures 6A-F are photographs of the proliferating rat hepatocytes cultured in HBM medium in the presence of HGF / SF and EGF as described herein and the Northern blots thereof.

Figure 6A is a photomicrograph of phase contrast of proliferating hepatocyte cultures coated with Matrigel on day 8 and photographed on day 18. The granular cytoplasm and appearance of typical bile canaliculi appear as bright lines between cells.

Figures 6B and 6C are low and high power electronic photomicrographs, respectively, of cells on day 18 in culture, 10 days after coating with Matrigel. Typical characteristics of the cytoplasm of hepatocytes are shown as endoplasmic reticulum lamellae wrapping around the mitochondria, microbodies with crystalline center, bile canaliculi ("c") (Figure 6B), abundant mitochondria ("M"), and glycogen ( "G") (both in Figure 6C).

Figure 6D is a photograph of a Northern blot showing the increased expression of albumin mRNA following the addition of Matrigel. Albumin mRNA is expressed in control cultures, immediately after isolation from the liver by collagenase perfusion and before culture. The expression is minimal on day 8 in the culture. On days 3 and 7 after the addition of Matrigel (bands marked with "+"), there was an increase in the expression of albumin mRNA. The expression of GADPH was used as an internal control.

Figure 6E is a photograph of a Northern blot showing the induction of cytochrome UBI mRNA in cultures treated with Matrigel on day 8, and exposed to phenobarbitol ("PB") 2 days later (day 10 of culture). The cells were harvested on day 15 of culture .. GAPDH expression was used as an internal control for loading the mRNA.

Figure 6F is a photograph of Northern blotting of cells further cultured with Matrigel (added at day 8 in culture). Cytokeratin 19, a marker of the bile ducts, expressed by proliferating hepatocytes, is suppressed by the addition of Matrigel and is not suppressed in control cultures that do not receive Matrigel. The 28S rRNA stained with ethidium bromide was used as an internal control.

Figures 7A-7E show the results of the studies showing the formation of the ductular / acinar structures in rat hepatocyte cultures set aside from the start of the culture between the layers of type I collagen gel in HBM in the presence of HGF / SF. "Figures 7A-7D are photographs of the crops on day 15.

Figure 7A is a phase contrast photomicrograph (100X) showing the appearance of the ductular structures surrounded by the collagen fibrils.

Figure 7B is a photomicrograph (100X) of the paraffin sections of the type I collagen gels of Figure 7A, stained with hematoxylin and eosin.

Figures 7C and 7D are electron micrographs of the cells surrounding the same lumen or lumen but at different sites around the lumen or lumen of one of the duct structures observed in Figure 7B. Figure 7C shows the cells with a morphology similar to the epithelium of the bile duct, with long parallel contacts joined by many desmosomes and with abundant intermediate filaments of keratin. Figure 7D shows the cells that resemble more the hepatocyte phenotype, with rough endoplasmic reticulum and mitochondria, densely stained secondary lysosomes and few filaments.

Figure 7E is a photograph of a Northern blot showing that the expression of cytokeratins 18 and 19 is increased in cultures with ductular / acinar structures, whereas albumin is only slightly expressed.

Figure 8 shows the photographs of the cultures of human stained hepatocytes, developed in HBM supplemented with HGF / SF and EGF, as described here, taken on day 1, 3, 5, 7, 10, 12 and 19.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a basal means of hepatocytes, designated HBM in the present, which allows the expansion, differentiation and long-term survival of the hepatocyte cell population, cell lines derived from hepatocytes such as HepG2, hepatic fetal epithelial cells, and Hepatic primary hepatocarcinoma cells in vi tro. In addition, the media of the present invention can be "used to culture pancreatic islet cells, renal tubular cells, Ito cells, epithelial cells of the small intestine, and a variety of cell lines, including MRC5, CaCo and 3T3 cells, as well as The HBM of the present invention maintains the metabolic pathways and synthesis functions, for example, the differentiation of mature adult hepatocytes, In addition, the HBM of the present invention, when combined with specific mitogens and extracellular matrix compositions. , hepatocytes can be excited to transdifferentiate into bile duct-like structures.The HBM of the present invention can be used for the culture of mammalian hepatocytes, and other cells, including, but not limited to those of human, rat origin. , of dog, pig, mouse and baboon.The HBM medium comprises appropriate levels of essential amino acids and non-essentials and ions and trace elements, buffers, vitamins, carbohydrates, lipids, proteins and hormones to function as a nutritive medium for the culture of mammalian cells in vi tro. In its broadest aspect, the invention characterizes a chemically defined basal medium, HBM, which by itself allows the long-term survival, differentiation and development of mammalian hepatocytes and other cells. Furthermore, in the presence of growth factors such as HGF / SF, or TGFα, as well as other mitogens, the cells that develop in the supplemented HBM have a faster population expansion and a faster clonal development. As further demonstrated below, the HBM of the present invention, when supplemented with HGF / SF, causes the formation of bile duct-like structures in conjunction with certain matrix constructs. As stated above, however, such growth factors are not required for specific differentiation patterns in primary crops, but they do accelerate development and population expansion if this is desired for a specific purpose. The HBM medium of the present invention comprises a chemically defined basal reserve medium (hereinafter referred to as "SM") designed for the culture of mammalian cells. The stock basal medium can preferably be constructed using the Dulbecco Modified Eagle Medium ("DMEM") as an ingredient, but the present invention is not so limited, as long as the formulations are within the guidelines described herein. Examples of other defined basal media that may be used in accordance with the present invention include, but are not limited to: Eagle Basal Medium (BME), DMEM / F-12 (DMEM and F-12 1: 1 vol: vol); Medium 199; Nutritive mixture F-12 (Ham); Nutritive mixture F-10 (Ham); Minimum Essential Medium (MEM); E half of Williams; and RPMI 1640, all of which are available from Gibco - BRL / Life Technologies, Inc., Gaithersburg MD, among others. Several versions of many of these media are available, and those that are particularly useful for building HBM include, but are not limited to: DMEM 11966, DMEM 10314, MEM 11095, Medium E 12251 from Williams, Ham F12 11059, MEM-alpha 12561 , and Medium 199 11151 (all available from Gibco-BRL / Li fe Technologies (catalog 1995-1996)). Therefore, for example, if L-arginine and / or D-glucose are already in the basal reserve medium in the necessary amounts, then little or no additional ingredient will have to be added as a supplement. Depending on the particular composition of the reserve basal medium, the SM is then supplemented, as more fully described herein, with D-glucose and / or D-galactose, nicotinamide, other micronutrients comprising amino acids and trace metals not present already in SM such as L-proline, L-glutamine, L-arginine, - L-ornithine, zinc, manganese, copper, and selenium, purified transferrin to which elemental iron or apo-transferrin binds in combination with gluconate of iron, hormones such as dexamethasone or insulin and a pH buffer such as HEPES (N- [2-hydroxyethyl] piperazine-N- [2-ethanesulfonic acid]). They can also be optionally imaged antibiotics such as penicillin - and streptomycin, an indicator of pH, alb-umina and / or dextran, essential fatty acids, alternative buffers, vitamins, osmotic agents, and other forms of trace metals. Typically, a basal medium will have a pH in the range of 6.5-8.2, preferably 7.0-7.7, and more preferably 7.2-7.5. Phenol red is a typical indicator added to help control pH. The SM and supplements for this comprise the HBM medium of the present invention. The HBM medium can then be optionally supplemented with one or more growth factors such as, for example, HGF / SF, EGF and TGFa if accelerated growth is desired. The simple carbohydrates D-glucose and / or D-galactose are added to the basal reserve medium to comprise HBM. If D-glucose and D-galactose are used, the sum of their total concentrations is preferably 8.0 g / 1 or less, but greater than .01 g / 1, taking into account the amount of D-glucose present in the medium basal reserve, if any. When only one of the D-glucose or D-galactose is used, the concentration is preferably 5.0-0.1 g / 1. For example, in the Example herein, the basal mixed DMEM stock medium that was used contained 2.0 g / 1 of D-glucose and no additional D-glucose was added. Then 2.0 g / 1 of D-galactose was added as a supplement. It has been shown that nicotinamide, another component of HBM maintains the differentiation of hepatocytes, improves the expression of cytochrome P450, and prolongs the survival of hepatocytes in conventional culture at 10-14 days. See, Rosenber, M.R. et al., In Vitro 18: 775 (1982) and Inoue, C. et al., Biol. Chem. 264 (9): 4747 (1989), the descriptions of which are incorporated by reference herein. Transferrin is a protein that binds to iron, which interacts with a transferrin receptor on the cell membrane. This serves to chelate and transport the iron ions. The transferrin preferentially used in the present invention is either 30% holo-transferrin saturated with iron and is completely unsaturated (apo-transferrin) and is combined with iron gluconate. Dexamethasone, a synthetic corticoid corticosteroid, has been shown to improve DNA synthesis induced by EGF as reported by Sand, T.F. and collaborators, Acta. Endocrinol. 109: 369 (1985), the description of which is incorporated by reference herein. As used in the present invention, dexamethasone can be any cortisol derivative such as, prednisone, predniß ilone, cortisol, hydrocortisone, and other derivatives. Insulin and insulin-like growth factors are required for glucose uptake, amino acid transport, and maintenance of multiple intermediary metabolic pathways. These effects help maintain differentiation and support proliferation. The inclusion of L-arginine in HBM, in the basal reserve medium, or supplementary to it, seems to be important because the hepatocytes in culture tend to lose their ability to synthesize arginine through the urea cycle. In the absence of L-arginine, hepatocytes in culture can not survive for long because they become unable to synthesize L-arginine, thereby blocking their protein synthesis. The use of D-galactose in addition to D-glucose is advantageous for HBM, because the combination gives the greatest growth potential on any single substance. As discussed above, HGF / SF, EGF, and TGFot are mitogens which, as shown below, show an increased proliferative effect when used together or alone according to the present invention. The above list of mitogens that can be used to supplement the HBM of the present invention is not exhaustive. It should be noted, however, that these mitogens are not required for the survival or differentiation of hepatocytes when they develop in HBM. The hepatocytes developed in HBM p'roli will fade at a slower rate than if mitogens were added. The insulin, EGF, HGF, and TGFα used in the media currently claimed may be either recombinantly produced, engineered, or purified from natural sources. The species can be, for example, human, bovine, equine, murine, porcine, or rat.

Various preferred reserve basal media - DMEM 11966, DMEM 10314, MEM 11095, and Medium E 12251 from Williams contain the following components per 1000 ml of sterile deionized water, as shown in Table I below.

TABLE I Compositions of Basal Reserve Medium (mg / l) EMEM DMEM MEM MEDIO E COMPONENT 11966 10314 11095 12251 DE WILLIAMS INORGANIC SALTS: CaCl2 (anhyd.) 200.00 200.00 200.00 200.00 CuS04- »5H20 0.0001 Fe (N03) - 9H20 0.10 0.10 0.0001 KCl 400.00 400.00 400.00 400.00 MnCl2 »4H20 0.0001 MgSO4 • (anhyd.) 97.67 97.67 97.67 97.67 NaCl 6,400.00 6,400.00 6,800.00 6,800.00 NaHC03 3,700.00 3,700.00 2,200.00 2,200.00 NaH2P04 * H20 125.00 125.00 140.00 NaH2PQ4 140.00 ZnS04 »7H20 0.0002 OTHER COMPONENTS D-Glucose 4,500.00 1,000.00 2,000.00 Glutathione 0.05 Methyl Linoleate 0.03 Red and Phenol 15.00 15.0.0 10.00 10.00 Sodium Piruvate 25.00 AMINO ACIDS L-alanine 90.00 L-arginine * HCl 84.00 84.00 126.00 L-arginine 50.00 L-asparagine «H20 20.00 L-aspartic acid 30.00 L-cysteine 40.00 L-cystine - 48.00 - - L-cystine »2HCl 63.00 63.00 31.00 26.10 L-glutamine 584.00 584.00 292.00 - L-glutamic acid - - - 50.00 Glycine 30.00 30.00 - 50.00 L-histidine »HCl« H20 42.00 42.00 42.00 L-histidine - - - 15.00 L-Isoleucine 105.00 105.00 52.00 50.00 L-leucine 105.00 105.00 52.00 75.00 L-lysine * HCl 146.00 146.00 73.00 87.50 L-methionine 30.00 30.00 15.00 15.00 L-phenylalanine 66.00 66.00 32.00 25.00 L-proline - - - 30.00 L-serine 42.00 42.00 - 10.00 L-threonine 95.00 95.00 48.00 40.00 L-triptof ano 16.00 16.00 10.00 10.00 L-tyrosine »2Na» 2H20 104.00 104.00 52.00 50.70 L-valine 94.00 94.00 46.00 50.00 VITAMINS: Ascorbic acid - - - 2.00 Biotin - - - 0.50 pantothenate D-Ca 4.00 4.00 1.00 1.00 Choline chloride 4.00 4.00 1.00 1.50 Ergocalciferol - - - 0.10 Folic acid 4.00 4.00 1.00 1.00 i-inositol 7.20 7.20 2.00 2.00 Menadione sodium bisulfate - - - 0.01 Niacinamide 4.00 4.00 1.00 1.00 Pyridoxine «HCl - 4.00 - - Pyridoxal * HCl 4.00 - 1.00 1.00 Riboflavin 0.40 0.40 0.10 0.10 a-tocopherol disodium phosphate 0.01 Thiamine »HCl 4.00 4.00 1.00 1.00 Vitamin A acetate 0.10 Vitamin B-.2 0.20 According to the preferred embodiment of the present invention, the following supplementary components in Table II are added to the basal reserve medium of Table I in a total volume of 1000 ml. The preferred amounts listed coincide with those described below in the Example for which the basal reserve medium was constructed from 455 ml of DMEM 10314 and 555 ml of 11966 to provide a glucose concentration of 2.0 g / 1, such that no additional D-glucose was added. It should be understood that when other basal reserve media are used, the optimal quantities of these components that are aggregated may vary.

TABLE II Additions to SM for HBM Preferred Quantity Concentration Range Units / 1 Units / 1 D-galactose 2.0 g 0.01-5.0 g * D-glucose 2.0 g ** 0.01-5.0 g * Nicotinamide 610.0 mg 1-3050 mg L-proline 30.0 mg ** 1-120 mg L-arginine 84.0 mg ** 1-150 mg L-ornithine 100 mg 1-500 mg human holo-transferrin (saturated 5.0 mg 0.1-100 mg with 30% Fe) h-insulin 5.0 mg greater than 10"11 M dexamethasone 1 x 10" 7 M 1012-10" 3 M ZnCl2 .544 mg ** 1-3000 μg MnS04 0.025 mg ** 1-250 μg ZnS04 »7H20 .750 mg ** 1-3000 μg CuS04 * 5H20 0.20 mg * * 1-1000 μg Selenium 5.0 μg 1-150 μg L-glutamine *** (additional to SBM) 5.0 mM 2.0-10.0 mM HEPES 20.0 mM 5-50 mM * If D-galactose and D-glucose are used, the upper limit is approximately 8.0 g / 1 or less, taking into consideration the quantities present in the basal reserve medium used, if any. The lower limit for when both or only one of D-glucose or D-galactose is used is approximately 0.01 g / 1 or greater. For example, in the Example herein, the mixed DMEM medium used contained 2.0 g / 1 of D-glucose without added additional amount., and 2.0 g / 1 of D-galactose. ** If it is not already in the basal medium of reserve SM. *** L-glutamine is a nutrient required in the present invention. Many basal reserve media are provided with included L-glutamine. However, it is well known to practitioners of the cell culture technique that L-glutamine will be degraded by oxidation, such that all L-glutamine will be degraded within a few weeks after manufacture. Therefore, the additional L-glutamine is added to the HBM medium of the present invention as described, to compensate for this.

Table III below lists the substances that may be optionally added to the HBM medium to optimize the development of the cells for specific purposes and for the specific species origin of the hepatocytes and the specific cell lines or malignant cells. Again, these amounts will vary depending on whether a particular component is already present in the basal reserve medium used.

TABLE III HBM Optional Additives Preferred Amount Interval of. Concentration Units / 1 Units / 1 Albumin * 2.0 '0-10.0 g Penicillin ** 100 U 0-2500 U Streptomycin ** 100 μg 0-2500 μg Sodium pyruvate 0.15 g 0-2.0 g Gamma-tocopherol 0.35 mg 0-3.5 mg Alpha-tocopherol 0.15 mg 0-2.5 mg Vitamin D3 0.20 mg 0-1.8 mg Dextran * 2.0 g 0-5.0 g Gluconate of 25.0 μg 0-100 μg Iron *** Lioleic acid 1.0 g 0-5.0 g Apo-tr nsferrin *** 5.0 mg 0-20.0 mg Retinol 0.05 mg • 0-2.0 mg Vitamin Bi2 0.J.5 mg 0-2.0 mg Ascorbic acid 3.0 mg 0-10.0 mg Choline chloride 1.5 mg 0.2-12.5 mg Biotin 0.75 0.2-15.0 mg * Dextran can be substituted by albumin. ** Other antibiotics may be used, such as, for example, gentamicin. *** Iron gluconate and apo-transferrin can be replaced by holo-transferrin saturated with iron.

In cases where accelerated growth is desired, various growth factors can be added to the HBM of the present invention. While HGF / SF, EGF and TGFa are currently preferred, it should be understood that other growth factors may also be used. The preferred amount of such growth factors will typically vary with the particular source used. The amounts listed herein are therefore necessarily limited to the particular sources used herein. In the following Example HGF / SF was preferably added to the HBM at 40 ng / ml; EGF was preferably added at 20 ng / ml; and TGFα was preferably added at 20 ng / ml. The following examples are included for illustrative purposes only, and are not intended to limit the scope of the invention.

EXAMPLE Materials and methods materials 344 male Fischer rats from Charles River, (Pennsylvania) were used for all experiments involving the isolation of rat hepatocytes. EGF and Matrigel (a mixture of matrix components derived from the EHS mouse tumor) were obtained from Collaborative Research (Waltham, MA). Tritiated thymidine was obtained from ICN Radiochemicals (Irvine, CA). Collagenase for the isolation of hepatocytes was obtained from Boehringer Mannheim (Indiannapolis, IN). Vitrogen (Celtrix Labs, Palo Alto, CA) was used for the construction of collagen gels. The general reagents were obtained from Sigma Chemical Co. (Saint Louis, MO). The HGF / SF used for these studies was the variant? 5. The ECL matrix was purchased from Upstate Biotechnology (Lake Placid, NY).

Isolation and Culture of Hepatocytes Rat hepatocytes were isolated by an adaptation of the two-step collagenase perfusion technique, with calcium, as shown by Kost, D.P. and collaborators, J. Cell. Physiol. 147: 274 (1991), the description of which is incorporated by reference herein. All type preparations were designed to obtain a population of pure hepatocytes. Conversely, to many other hepatocyte culture systems, the present invention does not utilize a co-culture of feeder cells or feeder cell conditioned media. After isolation of the hepatocytes, the cells were suspended in a medium used for coupling the cells to the culture plates. This medium was MEM (GIBCO 12570) with NEAA (GIBCO 11140), insulin 5 mg / ml, and gentamicin 5 μg / ml. The hepatocytes were plated on a single layer of collagen coating, as described below, and allowed to bind for 2 hours. Six-well accumulation plates (9.8 square centimeters per plate) of Corning were used. For experiments where hepatocytes were not going to be induced to proliferate, or where evaluations of the differentiated function were performed, the cells were plated at a density of 80,000 hepatocytes per square centimeter of surface area. For experiments aimed at inducing proliferation or genetic transduction with foreign DNA, the cells were plated at an initial density of 1,000 or 10,000 * hepatocytes per square centimeter of surface area. The plate placement medium was replaced with the HBM medium of the invention at 2 hours after the. cells were plated, and every 48 hours after this. Thymidine, growth factors and other ingredients were added at the time the medium change was required.

Human hepatocytes were isolated by an adaptation of the collagenase perfusion technique as described by Strom, S.C. and collaborators, Journal of National Cancer Institute 65 (5): 771-8 (1982), the disclosure of which is incorporated by reference herein. Cells were cultured as described above for rat hepatocytes. The collagen gels were prepared as described by Michalopoulos, G.K. and collaborators, Exp. Cell. Res. 2_4_: 70 (1975), the description of which is incorporated by reference herein. The dry coating of plates with collagen and Matrigel was also performed as specified by the manufacturer. Matrigel gels were prepared by the addition of 50 μl of Matrigel solution in 0T5 ml of medium directly on top of the coupled cells DNA synthesis was measured by uptake of tritiated thymidine into the precipitable material with trifluoroacetic acid (TCA), as described by Kost, DP et al. (1991), cited above.The collagen gels, where necessary, were digested with 2 mg of collagenase per ml of MEM medium used.Incubation was then carried out performed for 30 minutes at 37 ° C. The digested gels were treated with sodium hydroxide, followed by TCA to precipitate DNA, RNA and proteins as described by Kost, DP et al. (1991), cited above.

Composition of the HBM medium DMEM, HEPES, L-glutamine and antibiotics were purchased from GIBCO / BRL (Gaithersburg, MD). The ITS mixture (insulin, transferrin, selenium) was purchased from Boehringer Mannheim. All other additives were cell culture grade (Sigma). Unless indicated otherwise for the specific experiments, the basal reserve medium consisted of DMEM 11966 and DMEM 10314 mixed "to achieve a final D-glucose concentration of 2.0 g / 1. In this case, 445 were mixed. ml of DMEM 10314 with 555 ml of DMEM 11966 to achieve this concentration The formulas for these basal reserve media are described in Table I. The resulting mixed medium was then supplemented with: purified bovine albumin 2.0 g / 1, D- galactose 2.0 g / 1, L-ornithine 0.1 g / 1, L-proline 0.030 g / 1, nicotinamide 0.610 g / 1, ZnCl2 0.544 mg / l, ZnS04 * 7H20 0.750 mg / l, CuS04 * 5H20 0.20 mg / l, MnS04 0.025 mg / l, 5.0 mM glutamine, STI (rh-insulin 5.0 mg / l, human transferrin 5.0 mg / l [saturated with 30% diferric iron], selenium 5.0 μg / 1), dexamethasone 10 ~ 7 M, and HEPES 20.0 buffer, mM Penicillin and streptomycin were added at 100 U / 1 and 100 μg / 1, respectively The mixed basal HBM was sterilized by filtration to fed a 0.22 μm protein low binding filter system (Corning), stored at 4 ° C, and used within 4 weeks. Growth factors, as required, were added to the fresh HBM at the specified concentrations each time the medium was changed.

Retroviral Transfection and the Clonal Expansion Evaluation The hepatocytes were initially plaqued at loVcm2 and developed in HBM supplemented with HGF / SF (40 ng / ml) and EGF (20 ng / ml). After 68 hours the medium was replaced with the supernatant from the amphotropic retrovirus, replication deficient, packaged by CR? P (MFG approximately 5 x 105 units per ml) containing the E. coli β-galactosidase gene under a LTR promoter as described by Zitvogel, LH and collaborators, Hum. Gene. Ther. 5_: 1493 (1994), the description of which is incorporated by reference herein. Polybrene at 2 μg / ml was added. The supernatant was replaced after 18 hours with HBM which was supplemented with EGF at 20 ng / ml and HGF / SF at 40 ng / ml. The brief exposure with the virus-containing supernatant did not have an adverse effect on the survival or proliferation of the hepatocytes. At the indicated times, the cells were fixed with 0.5% glutaraldehyde in PBS for 10 minutes and developed with X-Gal substrate at 37 ° C for 16 hours. The transduced cells expressing the E. coli gene stained positively as shown in Figures 4G and 4F. The appropriate controls for each component were negative for X-Gal staining.

Transmission Electron Microscopy Samples for transmission electron microscopy (TEM) were fixed on culture plates for 1-1.5 hours in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% formaldehyde. The plates were then rinsed 2 times with 0.1 M sodium cacodylate buffer (pH 7.4) and 2 times with 0.1 M sodium cacodylate buffer containing 5% sucrose (pH 7.4). These were maintained in the sucrose buffer for 1-7 days, rinsed 2 times with the 0.1 M sodium cacodylate buffer (pH 7.4), and then post-set for 1 hour in 1% 0s04 in 0.1 M sodium cacodylate buffer The plates were then rinsed again in buffer, and the fixed and processed collagen gels were then cut into strips with a razor. The strips were then transferred to specimen glass jars, dehydrated through a graduated series of ethanol (25-100%) and two changes of propylene oxide, and infiltrated "with Epon-Araldite resin (BioTec, Texas). Various resin changes were made in 2 days, as the collagen gels tended to retain the propylene oxide.The collagen strips were embedded in a flat form and cured overnight at 60 ° C.

Analysis of Gene Expression by Northern Spotting Extraction of the RNA to such and the mRNA of Cul ti vos Total RNA was extracted from unwashed cell cultures using 2.0 ml RNAzol B (BioTec) per well and purified according to the manufacturer's guidelines. The concentration and purity of the RNA were determined by routine spectrophotometry. The size separation of 20 μg of RNA per band was completed on 1% denaturing agarose gels and transferred to nylon membranes (Amersham, Arlington Heights, IL) by the capillary method. After cross-linking under ultraviolet light, the membranes were hybridized overnight with the specific cDNAs (as indicated in the figures) that had been labeled with [α-32p] dCTP using a random primer Amersham kit. The membranes were subsequently washed under high stringency conditions and exposed to XAR films (Eastman Kodak, Rochester, NY) for 1-3 days. The quantification of RNA hybridization bands was performed by laser densitometry.

Sources of cDNA Probes The cDNA probes used to study gene expression were obtained as gifts, and are available at the request of the following sources; Citeroratina 8 of Dr. Norman Marceau (Laval University); Cytokeratin 14 from Dr. Dennis Roop (Baylor College of Medicine); Cytokeratin 18 by Dr. Robert Oshima (Foundation for Cancer Research of La Jolla); Cytokeratin 19 by Dr. Andre Royal (University of Montreal); TGFa (rat) from Dr. David Lee (University of North Carolina at Chapel Hill); EGF? R (rat) from Dr. Sheldon Earp (University of North Carolina at Chapel Hill); aFGF from the North American Collection of Species (American Type Culture Collection (ATCC) (catalog No. 78222), ATCC aFGF-R (catalog No. 65796), uPA from Dr. Jay Pegen (University of Cincinnati); UBI cytochrome from Dr. Steve Strom (University of Pittsburgh); ADNcs for albumin, α-fetoprotein, and transcription factor analysis were generated by Dr. Joe Locker (University of Pittsburgh).

RESULTS OBTAINED Role of the Components of the Media and Matrix Substrates on Cell Proliferation The complete description of the HBM medium is given above. To evaluate the relative importance of the different components of the media, several experiments were performed whose results are shown in Table IV (A, B and C) below. The components D-glucose, albumin, dexamethasone, transferrin and selenium, nicotinamide, and trace elements were individually subtracted from the complete composition of the HBM medium as shown in Table IV A. Total DNA per culture after 14 days of development is shown in this one. As you can easily see, the elimination of dexamethasone had the most dramatic effect, followed by the elimination of nicotinamide. In Table VI B, the development of cells achieved by day 14 is compared between the HBM medium containing diferric transferrin (saturated with iron) versus the unsaturated transferrin with iron. The addition of iron-containing differric transferrin (30% saturation) was found to be much more effective in promoting growth. The addition of elemental iron (ferrous sulfate, 0.1 μM) to the unsaturated transferrin failed to overcome the difference. Table VI C provides information on the relative effects of D-glucose, D-galactose and L-ornithine in the HBM medium. All of these three components are potential sources of energy for the cells. The complete cessation of development was perceived when all three components were removed. The addition of D-glucose alone restored most of the response, whereas the addition of D-galactose alone was less effective. Ornithine alone had minimal effect. It was found that the concentrations of 2 g per liter of each of albumin and D-glucose, are optimal, although the effects were not statistically different from 1 or 3 grams per liter in each case. The effect "of the complete elimination of these components is shown in Table IV A Table IV A. Effect of the Elimination of Specific HBM Components on the Growth of Hepatocytes μg / well DNA at time zero 13.90 ± 3.60 DNA at day 14 in HBM (with HGF / SF and EGF) supplemented with: + All components 84.90 ± 0.50 - Glucose 69.80 ± 2.90 - Albumin 68.70 ± 0.50 - Dexamethasone 13.70 ± 0.20 - (Transferrin and Selenium) 63.10 ± 2.00 - Nicotinamide 35.20 ± 1.70 - Elements in Traces 65.00 + 2.40 - All Components ~ 20.20 ± 4.30 B. Effects of Iron and Transferrin on the Growth of Hepatocytes DNA at time zero 9.21 ± 1.01 Transferrin diférrica 70.15 ± 1.21 (saturated with iron) Transferrin diférrica plus iron 1.83 ± 0.41 aggregate (5 μM) Transferrin poor in Iron 24.84 ± 4.30 Iron poor transferrin with 1.10 ± 0.90 iron added C. Effect of the Elimination of Glucose, Ornithine or Galactose Time zero 11.0 ± 0.4 Control (Gluc. +, Orn. +, Gal +) 59.0 ± 2.5 Gluc-, Orn. +, Gal.- 10.6 ± 0.8 Gluc-, Orn .--, Gal.- 14.4 ± 0.9 Gluc.-, Orn.-, Gal. + 44.2 ± 6.0 Gluc. +, Orn.-, Gal.- 59.0 + 4.0 Gluc. +, Orn. +, Gal.- 62.7 ± 1.4 G1UC. +, Orn.-, Gal. + "63.6 ± 0.3 The HBM components indicated were eliminated and the hepatitos developed in the modified medium for 14 days. The total DNA per culture in the microorganisms was measured at day 14 to evaluate the development of the growing cells. Time zero was the sample of the hepatocyte suspension that was inoculated into the plate immediately from the attachment of the cells. The data are expressed as the mean ± the standard error of three separate plates.

Hepatocytes Diffuse Entrance to Proliferation under the Influence of HG / SF, EGF, and TGFa Figures la and IB show the uptake of thymidine per μg of DNA, as well as the nuclear marker index of BRdU at different days in the culture in cells that develop in the presence of HGF / SF and EGF (40, 20 ng / ml respectively) as described above. As can be seen, most of the proliferation occurs at days 5-12. By day 15 the cultures were confluent and the DNA synthesis slowed down. The high rate of nuclear labeling during the "times of sustained proliferation indicates that proliferating cells are derived directly from mature hepatocytes, growth factors HGR / SF, EGF, TGFa, KGF (keratinocyte growth factor), SCF (totipotent cell factor), and aFGF (fibroblast growth factor, acid) were individually added to the HBM medium, from the aggregated growth factors, HGF / SF, EGF, and TGFa (shown in Figure 2 as "TGFa"). ") caused significant cell proliferation, as shown by the total amount of DNA per culture on day 15. When KGF, aFGF and SCF were added alone or in combination, they did not have a proliferative effect as shown in Figure 2. TGFa had a stronger proliferative effect than any other mitogens when added alone to the HBM medium.HGF / SF and EGF together had the strongest proliferative effect for a range of time given 15 days, than the one that had any other simple mitogens or combinations thereof. The addition of all the growth factors together had no more effect than the HGF / SF and EGF matched, as seen in Figure 2. The "detailed cell kinetics induced by HGF / SF and EGF alone or in combination, are shown in Figure 5. Total DNA per culture is shown as a function of time in culture.The highest amount of DNA accumulated at day 15 was observed with the combination of HGF / SF and EGF.DNA by culture on day 15 was 12 times aguel at time 0, reflecting the increase in the number of cells, HGF / SF and EGF were approximately equally potent, TGFa was only more mitogenic than HGF / SF or EGF alone.

Effect of Matrix Substrates Various matrix substrates promoted cell development in this system. The rat hepatocytes were cultured for 15 days in HBM supplemented with HGF / SF (40 ng / ml) and EGF (20 ng / ml) developed on various matrices, as seen in Figure 3. Cells were cultured as described above. Dry coating with type IV (mouse) collagens, type I (bovine), fibronectin and laminin, were equally effective in promoting cell growth, as assessed by measuring total DNA by culture at day 15. The coating dry with ECL (a commercial derivative of the EHS gel, UBI) had superior effects. Coating with type I collagen (commercial Vitrogen preparation) was the standard method used for the experiments, unless otherwise specified. The effect of matrix gels on the specific phenotypic conversions in these crops is further discussed below.

Phenotypic Changes of Hepatocytes During Proliferation The morphology of the proliferating cells (cultured in HBM as described above supplemented with 40 ng / ml of HGF / SF, and 20 ng / ml of EGF) varied at different times after stimulation of cell proliferation. From a normal hepatocyte morphology as seen in Figure 4A, proliferating cells in the first 4 days acquired long projections, assuming the phenotype typically described as due to the "scattering" effect of HGF / SF on the hepatocytes, as seen in Figure 4b, as described by Michalopoulos, GK and collaborators J. Cell. Physiol. 156: 43 (1993), the description "of which is incorporated by reference herein." Between days 6 and 8, the proliferating cells lost most of their cytoplasmic granules, the nuclei became less prominent, the projections The cells eventually began to develop as monolayer patches, eventually these patches were fused as the cells continued to grow to form a continuous layer as seen in Figure 4C. The electron microscopy examination observed in Figures 4D and 4E showed that Most of the typical characteristics of mature hepatocytes were absent.At the 15th day there were no lamellae of the endoplasmic reticulum surrounding the mitochondria, and there were no rosettes or glycogen peroxisomes.The bile canaliculi were absent. the intermediate filament bunches of geratine, the nuclei were angulated s with very prominent nucleoli. After confluence, the morphology gradually reverted to mature differentiated hepatocytes with endoplasmic reticulum sheets, mitochondria, glycogen, peroxisomes, bile canaliculi, etc., similar to agüellas in figures 6B and 6C.The clonal development of hepatocytes of proliferating rat is shown in Figures 4F and 4G.The hepatocytes were transfected on day three in culture with a replication-deficient retrovirus containing lac-Z gene under the influence of a viral LTR, and stained for expression of β-galactosidase Most of the single cells were stained positively on day 4 in culture (one day after transfection) as seen in Figure 4F. On the other hand, staining on day 10 and continuing until day 28, showed spots or patches of hepatocytes stained positively, consistent with the clonal development of the original transfected hepatocytes, as see Figure 4G. The percentage of lac-Z positive cells (approximately 20%) did not appear to change during culture.

Hepatocytes in Proliferation Express Altered Levels of Different Genes The expression of several specific genes in the proliferating hepatocytes was evaluated.

These included mRNA genes associated with the differentiation of hepatocytes (albumin, cytochrome UBI (labeled as P450 in Figure 5A)), the genes that code for the markers of cytokeratin (cytokeratins 14, 18 and 19) or related to the development of hepatocytes (urokinase (uPA), HGF / SF and its receptor c-met (marked as MET in Figure 5A), EGF (marked as EGFR) and TGFa and its receptor, FGF acid and its receptor, and TGFßl). These genes were studied by Northern blot analysis of RNA from cultures grown in the presence of either HGF / SF (40 ng / ml) and EGF (20 ng / ml) combined (Figures 5A and 5B) or with TGFa alone (FIG. 20 ng / ml) (data not shown). Total RNA was isolated from cultures on days 0, 6, 10, .15 and 21. No expression of AGF / SF or TGFβ1 mRNA was observed at any of the time points examined. As can be seen, albumin mRNA and cytochrome UBI were present at time zero, and subsequently decreased. Albumin mRNA was increased by day 21, time in which mRNA for α-fetoprotein (AFP) was also detected. The mRNA for cytokeratins 14, 18 and 19 was increased through the culture. There was a gradual increase in mRNA for aFGF and TGFa. The mRNA of the receptors for HGF / SF "(MET, Figure 5A) and aFGF (Figure 5B aFGFR), remained present throughout the culture time.The EGF receptor (EGFR) mRNA declined from day zero, but The expression of GAPDH mRNA was used as a reference gene for "home maintenance." There were dramatic increases in the expression of urokinase, as well as cytoglyratins 14 and 19. Some differences were observed from the previous pattern, in cultures that, instead of HGF / SF and EGF, were maintained in the presence of TGFa (20.0 ng / μril.) In these cultures the expression of albumin and the expression of the HGF / SF-receptor were better preserved during the proliferation, whereas AFP appeared earlier (Data not shown). Despite the differences observed in gene expression patterns, no morphological differences were observed between the cells that develop in the presence of TGFa or HGF / SF m EGF, once the confluence was reached.

Proliferating Hepatocytes Revert to Mature Hepatocytes Under the Influence of Matrigel or on Presence of Non-Parenchymal Cells or with Time in Culture When the Matrigel was coated on the day 8, there was a rapid appearance (within 2 days) of bile canaliculi and the organization of cells in similar structures in the form of a rope. The characteristics of these cells are shown in Figure 6A. As shown by electron microscopy in Figures 6B and 6C, these cells had typical markers of mature hepatocytes, including the envelopment of the endoplasmic reticulum around the mitochondria, the bile canaliculi, and the presence of glycogen. The mRNA preparations were made from cultures exposed to Matrigel for 10 days (days 8-18 in culture). The expression of albumin was compared between day zero in culture (immediately after perfusion with collagenase), on day 8 in culture (before the coating by Matrigel), and cultures on day 3 and 7 after coating with Matrigel, as shown in Figure 6D. The addition of Matrigel caused dramatic increases in the expression of albumin mRNA, in comparison to proliferating control cultures, in which it was minimally detectable. The effect "of phenobarbitol (PB) on the levels of cytochrome P450 mRNA UBI in the cultures treated with Matrigel, was also measured as shown in Figure 6E. Matrigel was added to the crops on day 8. PB was added 2 days later (day 10 of culture). The cells were harvested 5 days after the addition of PB (day 15 in culture). The addition of PB induced cytochrome mRNA mRNA only in cultures treated with Matrigel. The induction of this mRNA by phenobarbitol is typical of hepatocytes and does not occur in any other cell, as reported by Michalopoulos, G. et al., Science (Washington, DC) 193: 907 (1976), the description of which is incorporated by reference herein. Typically, hepatocyte cultures rapidly lose the ability to respond to PB. This finding is evidence that the addition of Matrigel to the cultures of the proliferating hepatocytes induces a mature hepatocyte phenotype, as is attested by the structure observed in electron microscopy, shown in Figures 6B and 6C. The expression of cytokeratin 19 (CK19) (Figure 6F), a bile duct marker expressed by proliferating hepatocytes before the introduction of "differentiation conditions, also ceased expression after the addition of Matrigel, as see Figure 5B DNA synthesis was measured in the cultures exposed to Matrigel, and there was a substantial decrease.To evaluate whether differentiation to mature hepatocyte morphology would require DNA synthesis, 20 mM hydroxyurea was added to the HBM medium. It has been shown that this (Michalopoulos, G. K. et al., Cancer Res. 3_8_: 1866 (1978), the description of which is incorporated by reference herein) inhibits the programmed synthesis of DNA in hepatocytes, by inhibition of ribonucleotide reductase Hydroxyurea was added to the cultures before coating with Matrigel and was maintained throughout the following period of 5 days. DNA synthesis decreased to 3.93% of control (without hydroxyurea) in the proliferating cultures maintained in the presence of Matrigel, and up to 6.27% of control (without hydroxyurea) in the cultures maintained in the presence of Matrigel. Although DNA synthesis was decreased to 6.27% of the control levels (+ Matrigel, without hydroxyurea), the conversion of proliferating hepatocytes to the morphology of mature hepatocytes was completely unaffected and involved the entire population.

HGF / SF (but not TGFa or EGF) Induces that the Hepatocytes in Proliferation Differentiate Ductular / Acinar Structures in Type I Collagen Gels Hepatocytes maintained between two layers of collagen gel retain su- morphology and differentiation for long periods of time as described by Michalopoulos, G.K. and collaborators, J. Cell. Physiol. 156: 443 (1993), the description of which is incorporated by reference herein. Hepatocytes maintained in collagen gel sandwiches in cultures with conventional media containing HGF / SF, undergo intense proliferation, form prominent projections, and eventually become organized in structures reminiscent of hepatic plaques. The behavior of hepatocytes maintained between two layers of collagen gel as previously described, but in the presence of HBM supplemented with either HGF / SF or EGF as described above, was examined. hepatocytes underwent typical phenotypic transitions as described above On the other hand, in hepatocytes in HBM supplemented with HGF / SF alone, after expansion of cells identical in appearance to the proliferating hepatocytes described above, multiple structures appeared in the form of ducts between on days 10 and 15. These became prominent and encompassed most of the cells present in the cultures, starting approximately from day 10 and day 15, most of the cells in the culture were accommodated in such ductular structures. The appearance of these structures is shown in Figure 7 A. Histological sections are shown in the Figur to 7B (light microscopy) and Figures 7C and 7D (electron microscopy). The structures had a ductular or acinar configuration. Some of the cells that surround these structures were very attenuated and had appearance by electron and light microscopy identical to the epithelium of the biliary duct. However, others are larger and more closely resemble the ductular hepatocytes described in previous studies of in vi vo models. The proliferation of the cells (data not shown) either under EGF or HGF / SF in the collagen gel sandwiches was much lower (<25% at the highest peak) than that observed in the cultures on plastic coated with collagen anhydrous. Most of the proliferation ceased by day 10 and duct-like structures appeared after cell proliferation had ceased (days 10-15). The ductular acinar structures were also noted in these cultures, when HGF / SF and EGF were combined, but were lower than with HGF / SF alone. As with the Matrigel coating, the addition of hydroxyurea to inhibit DNA synthesis (up to 5.1% inhibition of control) did not affect the formation of ductural structures (data not shown). Figure 7E shows that these cells express cytokeratin 19, which is characteristic of duct cells. (See, Sirica, AE, Prog. Liver Dis. _1_0: 63 (1992) and Sirica, A.K., Histol. Histopathology 10: 433 (1995), the descriptions of which are incorporated by reference herein. ). A small amount of albumin expression was also preserved, consistent with the presence of hepatocyte-like cells within the ductular structures, as seen in the Figure 7D. e noted that ductular cells maintain the expression of CK19 in the non-proliferating state (Figure 7F) in contrast to the cells that differentiate towards the mature hepatocyte line, 6 which stop the expression of this marker of the bile ducts.

Sustained Growth and Population Expansion of Human Hepatocytes in HGM, in Presence of HGF / SF and EGF Although cultures of human hepatocytes have not been characterized as extensively as those of the rat, available literature has shown that these cells also undergo a limited round of DNA synthesis after stimulation by growth factors and degenerate rapidly in culture. See, Ismail, T. et al., Hepatology 1_4: 1076 (1991), the description of which is incorporated by reference herein. The response of human hepatocytes to HGF / SF (40 ng / ml) and EGF (20 ng / ml) in HBM medium was also studied. Results similar to those described for rat hepatocytes were found in the primary cultures of human hepatocytes, as seen in Figure 8. Human cells begin to proliferate rapidly on day 3-4 in culture, and reach confluence by on the 19thAs stated above, there is a current need for a medium that allows hepatocytes that are cultured in vi tro to expand as a cell population. The HBM of the present invention allows such expansion and therefore allows much necessary research of the proliferating hepatocytes. The present invention will also be useful in numerous other applications described below. For example, all current methods in liver-directed gene therapy, which achieve stable long-term expression of the transferred genes, require actively dividing cells during the initial transfection. The normal liver has only 1 in 20,000 growing hepatocytes in the S phase on any particular day. In order to increase cell proliferation, a large portion (2/3) of the patient's liver must be removed. Subsequently, the source of the gene carrier directed to the liver is intravascularly injected. The alternative to this drastic measure is to remove a piece of liver (10%), grow the hepatocytes, transfer them in culture, and then reinfuse the cells back to the liver. The latter method, while being safer, less expensive and better controlled than the former, is currently hampered by the lack of culture media such as HBM, which allow for prolonged proliferation, essentially clonal expansion of all cultured cells, and the long-term viability of the cells. The design of the current devices of extracorporeal liver, based on bioartificial hepatic tissue, which includes cellular elements, rely on transformed hepatocytes (tumors) or animal hepatocytes. Animal cells do not survive more than a few days in the bioreactors that are used, and do not proliferate, making it necessary to generate bioreactors more frequently and in close proximity and maintain all the necessary facilities, materials, and animal donors in anticipation of need. In addition, animal cells can have several undesirable aspects that make them clinically difficult or dangerous to use. Also, the tumor cells used in these devices have the risk of leakage of cells to the patient and therefore could potentially give rise to tumors in the patient. The HBM medium of the present invention will allow the production of bioreactors containing animal and / or human hepatocytes and / or other cells, and will provide the unique characteristics of increasing cell numbers, while maintaining long-term viability and complete differentiation Each reactors could be useful for the. drug manufacturing, drug metabolism, toxicology studies, and complex biological studies. Another potential use for the HBM medium of the present invention is an autotransplant of hepatocytes. Hepatocyte transplantation is a relatively new procedure to treat patients with liver disease in the final stage. The method relies on the use of donor organs, which are not used for organ transplantation. The autotransplant has the advantage of not requiring immunosuppressive drugs, but it is not practical if it is not possible to expand the number of cells and keep them alive for a sufficiently long time to place them back inside the donor.The liver explants in the final stage could also provide a source of donor hepatocytes, under selected conditions, including encapsulation or genetic manipulation, notwithstanding the source of hepatocytes, the expansion of the number of cells in the long-term culture and the reimplantation (within the spleen, the portal system, the peritoneum, renal capsule, etc.), it does improve the effective synthetic and detoxification processes, for prolonged periods of time.The limiting point is the lack of hepatocytes.HBM has the potential to eliminate this lack of cells, and with this expand dramatically the number of patients who could be treated for liver failure. Similarly, autotransplantation or heterotransplantation with other cells developed in the media of the present invention could expand the number of patients and the arrangement of possible treatments with biocellular therapies. The HBM of the present invention may also be useful for drug and chemical product testing procedures Virtually all drugs, chemicals, and other manufactured products must be tested for mutagenicity or toxicity in hepatocyte cultures, as part of of the FDA, USDA, NIOSH, and EPA regulations, these tests are currently performed on rat hepatocytes in short-term culture due to lack of long-term viability or significant proliferation. ) will extend the trial period (longer-term viability) (2) allow longer periods of exposure to lower concentrations, (3) allow for the observation of mutagenicity and toxicity on proliferating hepatocytes, and (4) will make possible a supply of human hepatocytes which could be used in other toxicological investigations, although the invention has been described in detail for purposes of illustration, it should be understood that such details are solely for that purpose, and that variations may be made herein by those skilled in the art without departing from the spirit and scope of the invention, except as this may be limited by the claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (43)

1. A cell culture medium capable of expanding the hepatocytes in vi tro without serum, characterized the porgue medium comprises: nicotinamide, iron-bound transferrin, insulin or insulin-like growth factors, glucocorticoid steroid, trace metals, carbohydrates selected from the group consists of glucose and galactose, arginine, proline and glutamine, in sufficient amounts to increase the total number of cells in culture above the total number of viable cells introduced into the culture.

2. A cell culture medium capable of expanding the hepatocytes in vi tro without serum, characterized in that it comprises: (a) basal reserve medium for mammalian cell culture; and (b) nicotinamide, iron-bound transferrin, insulin or insulin-like growth factors, glucocorticoid steroid trace metals, carbohydrate selected from the group consisting of glucose and galactose, arginine, proline, and glutamine, when either of these is not present. content in the reserve basal medium in sufficient quantities to increase the total number of culture cells, above the total number of viable cells introduced into the culture.

3. The culture medium according to any of claims 1 or 2, further characterized in that it comprises one or more of the additional growth factors in the cell culture medium according to claim 1 or 2, said growth factor functioning as a mitogen for the cells.

4. The cell culture medium according to claim 1, further characterized by comprising one or more of the following: growth factors that function as a mitogen for the cells; albumin, 0-10 g / 1; sodium pyruvate 0-2.0 g / 1; gamma-tocopherol, 0-3.5 mg / l; alpha-tocopherol, 0-2.5 mg / l; vitamin D3, 0-1.8 mg / l; dextran, 0-5.0; linoleic acid 0-5.0 g / 1; apotransferrin, 0-20.0 mg / l and iron gluconate, 0-100 μg / 1; retinol, 0-2.0 mg / l; vitamin B? 2, 0-2.0 mg / l; ascorbic acid 0-10.0 mg / l; Choline chloride, 0.2-12.5 mg / L; biotin, 0.2-15.0 mg / l.

5. The culture medium according to claim 2, further characterized by including one or more of two following: growth factor which functions as a mitogen for the cells; albumin, 2.0 g / 1; sodium pyruvate, 0.15 g / 1; gamma-tocopherol, 0.35 mg / l; alpha-tocopherol, 0.15 mg / l; vitamin D3, 0.20 mg / l; dextran, 2.0 g / 1; linoleic acid, 1.0 g / 1; apotransferrin, 5.0 mg / l and iron gluconate, 25.0 μg / l; retinol, 0.05 mg / l; vitamin B? 2, 0.15 mg / l; ascorbic acid, 3.0 mg / l; Choline chloride, 1.5 mg / L; biotin 0.75 mg / l.

6. The culture medium according to claim 3, characterized in that one or more of the additional growth factors are selected from the group consisting of hepatocyte growth factor, epidermal growth factor, and trans-forming growth factor alpha.

7. The culture medium according to claim 2, characterized in that the reserve basal medium is selected from the group consisting of DMEM, MEM, Williams E-medium, BME, DMEM / F-12, Medium 199, nutritional mixture F-12 (Ham), nutritious mixture F-10 (Ham), and Medium 1640 of RPMI.

8. The culture medium according to claim 7, characterized in that the basal medium is DMEM.

9. The culture medium according to claim 8, characterized in that DMEM contains 2.0 g / 1 of D-glucose.

10. The culture medium according to any of claims 1 or 2, characterized in that the trace metals comprise zinc, manganese, copper, and selenium.

11. The culture medium according to claim 10, characterized in that the trace metals further comprise ZnCl, ZnS0 and CuS04.

12. The cell culture medium according to any of claims 1 or 2, characterized in that the glucocorticoid steroid is selected from the group consisting of cortisol, cortisol derivative such as prednisone, prednisolone, and hydrocortisone, and dexamethasone.

13. A cell culture medium capable of expanding the intravenous hepatocytes, characterized in that it comprises a basal reserve medium with the following components: calcium chloride (anhydrous), 200.00 mg / l; ferric nitrate nonahydrate (Fe (N03) 3- • 9H20), 0.10 mg / l; potassium chloride 400 mg / l; magnesium sulfate (anhydrous), 97.67 mg / l; sodium chloride (NaCl), 6,400.00 mg / l; sodium hydrogen carbonate (NaHCO3) 3,700.00 mg / l; hydrous sodium dihydrogen phosphate (NaH2P04 »H20), 125.00 mg / l; dihydrochloride (2 HCl) of L-cystine, 63.00 mg / L; glycine, 30.00 mg / l; hydrated L-histidine hydrochloride (HC1- »H20), 42.00 mg / l; L-Isoleucine, 105.00 mg / L; L-leucine, 105.00 mg / l; L-lysine hydrochloride, 146.00 mg / l; L-methionine, 30.00 mg / l; L-phenylalanine, 66.00 mg / l; L-serine, 42.00 mg / l; L-threonine, 95.00 mg / l; L-tryptophan, 16.00 mg / l; L-tyrosine, 2Na »2H20, 104.00 mg / l; L-Valine, 94.00 mg / L; D-calcium pantothenate, 4.00 mg / l; Choline chloride, 4.00 mg / l; folic acid, 4.00 mg / l; i-inositol, 7.20 mg / l; pyridoxine hydrochloride, 4.00 mg / l; pyridoxal hydrochloride, 4.00 mg / l; riboflavin, 0.40 mg / l; thiamine hydrochloride, 4.00 mg / l; D-galactose, 0.01-5.0 g / 1; D-glucose, 0.01-5.0 g / 1; nicotinamide 1-3050 mg / l; L-proline, 1-120 mg / l; L-arginine, 1-150 mg / l; L-ornithine, 1-500 mg / l; holotrans human ferrine (saturated at 30% with Fe), 0.1-100 mg / l; h-insulin greater than 10"11 M; dexamethasone 10" 12-10-13 M; zinc chloride (ZnCl2), 1-3000 μg / 1; manganese sulfate (MnS0), 1-250 μg / 1; zinc sulfate heptahydrate (ZnS04 »7H20), 1-3000 μg / 1; copper sulphate pentahydrate (CuSO4 * 5H20), 1-1000 μg; selenium, 1-150 μg / 1; L-glutamine, 2.0-10.0 mM; HEPES, 5-50 mM.

14. The culture medium according to claim 11, characterized in that it is found in the following components in the specified amounts: purified bovine albumin; D-glucose, 2.0 g / 1; D-galactose, 2.0 g / 1; L-ornithine, 0.1 g / 1; L-proline, 0.030 g / 1; nicotinamine, 0.610 g / 1; zinc chloride (ZnCl2), 0.544 mg / l; zinc sulfate heptahydrate (ZnS0 * 7H20), 0.750 mg / l; copper sulphate pentahydrate (CuS04 * 5H20), 0..20 mg / l; manganese sulfate (MnS0), 0.025 mg / l; glutamine 5. 0 mM; ITS rh-insulin, 5.0 mg / l; human transferrin (saturated at 30% with diferric iron), 5.0 mg / l; selenium, 5.0 μg / 1; dexamethasone 10 ~ 7 M; and HEPES 20.0 mM buffer.

15. A mixture of the components, characterized in that when they are added to the liquefied form the cell culture medium according to any of claims 1 or 2.

16. A method for the manufacture of cell culture medium according to any of claims 1 or 2, characterized in that it comprises the addition of dry or anhydrous components to the liquid to form the culture medium.

17. A composition, characterized in that it comprises hepatocytes in culture medium according to any of claims 1 or 2.

18. A method for expanding in vitro hepatocytes, characterized in that it comprises introducing the hepatocytes into the cell culture medium according to any of claims 1 or 2, and cultivating said hepatocytes introduced into culture medium.

19. A method for expanding hepatocytes in vi tro, characterized in that it comprises the introduction of the hepatocytes into the cell culture medium according to any of claims 1 or 2, and allowing cell proliferation to occur by culturing the hepatocytes in said cell. medium.

20. A method for altering the phenotype of a hepatocyte to a less differentiated state in vi tro characterized the method because it comprises the introduction of the hepatocytes into the cell culture medium according to any of claims 1 or 2, by culturing the hepatocytes introduced into the cell culture medium. the medium, to allow cell proliferation to occur, to allow cell proliferation for a sufficient time to produce cells that are less differentiated than the introduced hepatocytes.

21. A method for the production of hepatocytes in vi tro, characterized in that it comprises the introduction of the hepatocytes in the cell culture medium according to any of claims 1 or 2, the culture of the hepatocytes at a less differentiated stage, by allowing cell proliferation occurs, for a sufficient time, and causing less differentiated cells to develop the characteristics of introduced hepatocytes, where the development of the characteristics of the hepatocytes is originated by a method selected from the group consisting of the addition of the material of extracellular matrix and allowing less differentiated cells to reach confluence.

22. The method according to claim 21, characterized in that the matrix comprises one or more of fibronectin, collagen, laminin, and polylysine.

23. The method according to claim 21, characterized in that the matrix comprises entactin, laminin, and type IV collagen.

24. A method for the production of clonal development of cells derived from hepatocytes, in vi tro, the method is characterized in that it comprises the introduction of the hepatocytes into the medium according to any of claims 1 or 2, and allowing proliferation to occur. cellular and clonal development, through the culture of hepatocytes in the medium.

25. A method for the formation of ductular or acinar structures from in vitro hepatocytes, characterized in that the method comprises the introduction of the hepatocytes into the cell culture medium according to any of claims 1 or 2, the culture of the hepatocytes. at a less differentiated stage, by allowing cell proliferation to occur for a sufficient time, and causing the less differentiated cells to develop the characteristics of the introduced hepatocytes, where a matrix is used to develop the characteristics, and the structures are formed by the addition of one or more growth factors to the cells on the matrix.

26. A method for expanding the cells in vi tro, characterized in that it comprises introducing the cells into the cell culture medium according to any of claims 1 or 2, and culturing the introduced cells into the medium, wherein the cells are selected from the cell. a group consisting of pancreatic islet cells, renal tubular cells, hepatic pericytes, and epithelial cells of the small intestine.

27. A method for expanding the cells in vi tro, characterized in that it comprises introducing the cells into the cell culture medium according to any of claims 1 or 2, and allowing cell proliferation to occur by culturing the cells in said cell. medium, wherein the cell is selected from the group consisting of pancreatic islet cells, renal tubular cells, hepatic pericytes, and epithelial cells of the small intestine.

28. A method for altering the phenotype of a cell to a less differentiated state in vi tro, characterized by comprising the introduction of the cells into the cell culture medium according to any of claims 1 or 2, by culturing said cells introduced into the medium , to allow cell proliferation to occur, allowing cell proliferation for a sufficient time to produce cells that are less differentiated than the introduced cells, wherein the cell is selected from the group consisting of pancreatic islet cells, renal tubular cells, hepatic pericytes, and epithelial cells of the small intestine.

29. A method for the production of cells in vi tro, characterized in that it comprises the introduction of the cells in the cell culture medium according to any of claims 1 or 2, cultivating the cells at a less differentiated stage allowing cell proliferation to occur for a sufficient time, and causing the less differentiated cells to develop the characteristics of the introduced cells, where the development of the cellular characteristics is originated by a method selected from the group consisting of the addition of extracellular matrix material and allowing the less differentiated cells reach confluence, wherein the cell is selected from the group consisting of pancreatic islet cells, renal tubular cells, hepatic pericytes, and epithelial cells of the small intestine.

30. A method for the production of clonal growth of cells derived from in vi tro cells, the method is characterized by comprising the introduction of the cells into the medium according to any of claims 1 or 2, and allowing cell proliferation to occur and clonal development by culturing the cells in the medium, wherein the cell is selected from the group consisting of pancreatic islet cells, renal tubular cells, hepatic pericytes, and epithelial cells of the small intestine.

31. A method of expanding the cells in vitro, characterized by comprising the introduction of the cells into the cell culture medium according to any of claims 1 or 2, and cultivating the cells introduced into the medium, wherein the cell is selected. of the group consisting of the cell lines MRC5, CaCo and 3T3.

32. A method for expanding the cells in vi tro, characterized in that it comprises introducing the cells into the cell culture medium according to any of claims 1 or 2, and allowing cell proliferation to occur by culturing the cells in said medium , wherein the cell is selected from the group consisting of the MRC5, CaCo and 3T3 cell lines.

33. A method for the production of clonal growth of cells derived from in vi tro cells, the method is characterized in that it comprises introducing the cells into the medium according to any of claims 1 or 2 and allowing cell proliferation to occur and Clonal development by culturing the cells in the medium, wherein the cell is selected from the group consisting of the MRC5, CaCo and 3T3 cell lines.

34. A cell produced by the process according to claim 18.

35. A pharmaceutical composition, characterized in that it comprises the cells according to claim 34.

36. A method for the production of a recombinant cell expressing a heterologous gene, characterized by the porgue method, transforms any of the cells according to claim 34 with nucleic acid capable of expressing said gene in the cell.

37. A method for using the cells according to claim 36, characterized in that it comprises infusing the cells into a patient and allowing the gene to be expressed.

38. A method for using cells according to claim 34, characterized in that it comprises infusing the cells in a patient.

39. A method for the transplantation of hepatocytes, characterized in that it comprises the introduction of the hepatocytes according to claim 18, in a patient.

40. A method for manufacturing a gene product, characterized in that it comprises the culture of the cells according to claim 36 and the recovery of the gene product.-

41. A method for testing a drug, characterized in that it comprises introducing the drug to the cells according to claim 34 and evaluating the effect of the drug.

42. Hepatocytes in a culture medium, characterized in that it comprises arginine and transferrin chelated with iron.

43. A method for maintaining differentiated hepatocytes, characterized in "the method comprising the culture of the hepatocytes in the cell culture medium according to any of claims 1 or 2.