No abstract

The culture conditions found to result in stable proliferation of normal rat hepatocytes are: (i) subconfluent cell densities; (ii) serum-free medium; (Wi) hormonally defined medium containing epidermal growth factor, insulin, glucagon, prolactin, and other growth factors; and (iv) substrata of liver extracellular matrix depleted of growth inhibitors. Serum was found deleterious to parenchymal cells: it was inhibitory to the expression of liver-specific functions, cytostatic to parenchymal cells at all seeding densities, and cytotoxic to them at low seeding densities. These studies emphasize the relevance of synergies in the influences of hormones and extracellular matrix in regulating hepatocellular physiology.Past efforts to produce long-term proliferation of adult hepatocytes in culture have met with limited success (1-8 Substrata. Cells were plated on one of the following substrata: (i) Tissue culture plastic. Sixty-millimeter tissue culture dishes (Falcon). (ii) Type I collagen gels. Sixty-millimeter tissue culture dishes (Falcon) were coated with type I collagen gels prepared from rat tail tendons (10). (ifi) Normal rat liver biomatrix. Livers from normal Sprague-Dawley rats were utilized to prepare biomatrix by the methods of Reid (12). The biomatrix was pulverized into a fine powder with a Spex-Mill liquid nitrogen pulverizer (Spex-Mills, Metuchen, NJ), thickly smeared onto 60-mm Petri dishes (Falcon) and then sterilized by 'y irradiation (cesium-135) at 10,000 rads (100 grays). Just before use, the plates were rinsed with serum-free RPMI 1640 medium supplemented with penicillin at 200 units/ml and streptomycin at 200 pug/ml. (iv) Rat regenerating liver biomatrix. Five days after partial hepatectomy (18), livers were used for biomatrix preparation (12). (v) Normal rat liver biomatrix prepared by low-salt extraction. Biomatrix from normal rat liver was prepared by extracting the tissue with 1.0 M NaCl and omitting the initial wash in distilled water, prepared as described (11).Morphological Studies. Cultures of hepatocytes were plated under various conditions and maintained for 1-2 weeks. They were examined by using a Nikon inverted phase-contrast microscope. Cultures were evaluated by a number of investigators independently. Representative cultures were selected and photographed with phase-contrast microscopy.[3H]Thymidine Incorporation. At 1411The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody pioduced against ligandin, bear entirely sinlilar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was showh to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.ligandin is a cytoplasmic protein found in abundance in the liver of rat, man, and other species. This protein is capable of binding noncovalently a large number of compounds, which includes bilirubin, heme, benzyl penicillin, certain steroids, and such dyes as bromosulfophthalein and indocyanine green (1, 2). Phylogenetic, ontogenetic, induction, and competition studies support the hypothesis that ligandin is a major determinant of the net flux of organic anions from plasma into the liver (3-7). Fractionation of the protein on the basis of any one of its binding activities has resulted in apparently identical, highly purified preparations (7-9); Thus, the term, ligandin (2), is synonymous with that of azo-dye carcinogenbinding protein (8), corticosteroid binding I protein (9), and Y protein (7)$, §.The glutathione S-transferases (EC 2.5.1.18) from rat liver (iO-i3) have sevieral physical properties in common with rat liver ligandin (2). When crude liver extracts were subjected to filtration on Sephadex G-75, the fraction containing ligandin also served as a source of enzymatic activity for the conjugation of glutathione (GSH) with 1,2-dichloro-4-nitrobenzene (14). However, subsequent fractionation resulted in removal of alriost all GSH transferase activity with this substrate despite virtually complete recovery of ligandin (t, 15).Since four of the GSH transferases of rat liver (transferases A, B; C, and E) have been purified to homogeneity (11-13), (w/v) and the precipitate was removed by centrifugation after an additional 4 hr at 4°. The superratant fluid was used directly for enzyme assays with 1,2-ehloro-4-nitrobenzene. The precipitate was washed twice with phosphate-buffered saline at pH 7.4, containing 2%o polyethylene glycol. The residue was dissolved in 0.5 M NaOH, and absorbance at 280 nm was determined.

A new procedure is introduced for the isolation of connective tissue fibers, called biomatrix, containing a significant portion of the extracellular matrix (basement membrane components and components of the ground substance) . Biomatrix isolated from normal rat liver contains >90% of the tissue's collagens and all of the known collagen types, including types I and III and basement membrane collagens. The purified collagenous fibers are associated with noncollagenous acidic proteins (including fibronectins and possibly small amounts of glycosaminoglycans) . Procedures are also described for preparing tissue culture substrates with these fibers by either smearing tissue culture dishes with frozen sections or by shredding the biomatrix into small fibrils with a homogenizer. The biomatrix as a substrate has a remarkable ability to sustain normal rat hepatocytes long-term in culture. The hepatocytes, which on tissue culture plastic or on type I collagen gels do not survive more than a few weeks, have been maintained for more than 5 mo in vitro when cultured on biomatrix. These cells cultured on rat liver biomatrix show increased attachment and survival efficiencies, longterm survival (months), and retention of some hepatocyte-specific functions.Despite numerous advances in cell culture procedures (2, 3,14,16,18,24,35), the culture of differentiated epithelial cells, particularly from normal tissues, has remained especially difficult. We have proposed that the shortcomings of cell culture techniques are primarily that cells are isolated from the extracellular matrix and from association with other cell types with which they may be interdependent (30). Culture methods, such as organ culture or the culture oftissue fragments, which retain tissue architecture, preserve the differentiative state of the cells, whereas clonal cell cultures usually undergo a dedifferentiative process (16,40). Thus, to culture differentiated cells it seems logical to ascertain the critical variables of the tissue matrix and to evolve culture procedures dependent upon them.In a previous paper, we presented techniques that we refer to as "socio-cell culture techniques" (30) and that are, in essence, attempts to simulate some of the cell-cell relationships of the tissue matrix relevant to epithelial cells . The techniques we described involve the culturing of epithelial cells on substrates of reconstituted basement membrane and in medium THE JOURNAL OF CELL BIOLOGY " VOLUME 87 OCTOBER 1980 255-263 © The Rockefeller University Press -0021-9525/80/10/0255/09 $1 .00 supplemented with hormones, serum, and with conditioned medium from feeder layers of primary cultures of fibroblasts . In this article, we present new techniques that have superseded the earlier ones. They include the isolation of connective tissue fibers called biomatrix, representing a significant portion ofthe in vivo extracellular matrix (basement membranes and ground substance), and techniques using these fibers as substrates for cultures of differentiated cells . Our lon...