Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix.

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.

A Mathematical Model of Liver Cell Aggregation In Vitro

“…Data on the rates of proliferation and death of hepatocytes in culture are limited. Enat et al (1984) report that the ratio of the number of cells on day 7 of culture compared to that on day 1 is 1.0-2.9 (depending on the type of ECM and culture medium used), whilst Thomas et al (2006) found around 10 % of cells died between 24 and 48 hours in culture. Since the timescale of interest for aggregation is around 1 day, this suggests that cell proliferation is not the main cause of cluster formation.…”

?3?1-integrin as a critical mediator of the hepatic differentiation response to the extracellular matrix

“…Indeed, albumin mRNA and other liverspecific products decline markedly when hepatocytes are isolated from the liver and cultured on a plastic substratum, whereas albumin and other liver-specific mRNAs continue to be expressed at high levels when primary hepatocytes are cultured on collagen ECM gels. 2,3,5 We therefore investigated whether albumin mRNA induction was perturbed when the ␣ 3 antisense cell lines are shifted to a collagen gel substratum. We cultured the ␣ 3 -AS2 and ␣ 3 -AS7 antisense cell lines and two sense-strand control lines on collagen gels for 4 days.…”

“…42 Serum-free medium was as described by Enat et al 3 Media usually contained penicillin and streptomycin at 100 units/mL. For cell-surface labeling, confluent monolayers were seeded and cultured for 2 days on either tissue culture plastic or gels of rat tail collagen type I (Collaborative Research, Boston, MA), which were prepared as described previously.…”

“…For example, early hepatocyte differentiation begins as foregut endodermal cells enter the new collagenous environment of the surrounding mesenchyme, 1 and the role of the ECM in adult hepatocyte differentiation is well established. [2][3][4][5][6][7][8] Other examples of ECMpromoted differentiation include the differentiation of keratinocytes, 9 gastrulation in Pleurodeles embryos, 10 and the epithelial conversion of kidney mesenchyme 11 (for review, see Adams and Watt 12 ). The ECM is present in all animals from the earliest stages of development and is composed of a mixture of proteins and proteoglycans such as collagens, laminin, and fibronectin.…”

“…3,[6][7][8][26][27][28][29][30][31] Freshly isolated rat hepatocytes can bind to collagen via ␣ 1 ␤ 1 -integrin, 32 as shown by the ability of a monoclonal antibody to the ␣ 1 subunit to block binding to native type I collagen. 33 Hepatocyte binding to fibronectin, specifically at the amino acid sequence RGD, is mediated by ␣ 5 ␤ 1 -integrin.…”