Connective tissue biomatrix: its isolation and utilization for long-term cultures of normal rat hepatocytes.

Abstract

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...

Primary culture, cellular stress and differentiated function

“…Maintenance of normal in vivo levels of constitutively synthesized tissue specific proteins is significantly enhanced by culturing these cells on or within specific biomatrices such as collagen gels [53,54], or matrices from other natural tissue glycoproteins such as fibronectin [SO]. Complex biomatrices have also been employed, as for example, that secreted by bovine corneal epithelial cells [41], or isolated from rat liver [55]. However, liver parenchymal cells are able to synthesize their own collagen biomatrix in culture [56].…”

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis

“…Preparation of Liver Biomatrix. Biomatrix was isolated as previously described, 20 with modifications. Samples of livers from control and CCl 4 -treated rats were homogenized briefly in ice water.…”

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis

“…Biomatrix was isolated as previously described, 20 with modifications. Samples of livers from control and CCl 4 -treated rats were homogenized briefly in ice water.…”