Characterization of a digestase preparation and its use to obtain primary cell cultures

Abstract

The action of the preparation Digestase on several protein and synthetic substrates is examined, as well as the effect of a number of proteinase inhibitors on this enzyme preparation. Digestase has been found to exhibit considerable trypsinlike and collagenolytic activity. Experience with the use of Digestase to obtain isolated adenohypophyseal, hepatic, and cardiac cells is described, and some parameters of the functional activity of these cells in the course of their cultivation are evaluated. [4,5,8] among other research purposes. To obtain adenohypophyseal and hepatic cell cultures, use is usually made of trypsin and collagenase, respectively [5,8]. The proteolytic activity of trypsin against hepatic cells has been shown to be so high that it damages their cytoplasmic membranes irreversibly even in very low (0.001%) concentrations [3]. For the isolation of cardiac cells, trypsin or collagenase is employed [13]. At present, there is a distinct need for expanding the range of enzymes that can be utilized to produce primary cell cultures. In this article we describe our experience with the use of Digestase for the isolation of adenohypophyseal, hepatic, and cardiac ceils, and also assess some parameters of the functional activity displayed by the cells obtained. Because Digestase is a composite preparation of enzymes with both collagenolytic and trypsinlike activity [11,14], it is used to obtain adenohypophyseal and hepatic cells either alone or in combination with lima bean trypsin inhibitor (LBTI) or with fetal bovine serum (FBS) as an inhibitor of proteolytic activity. MATERIALS AND METHODSDigestase, a composite preparation of collagenolytic proteinases derived from the hepatopancreas of the crab Paralithodes camtschatica, was made by the method described in the European patent EP 0402 321 AI.Proteolytic activity was assayed by in vitro hydrolysis of 14C-acetylated acid-insoluble type I collagen [10], type IV collagen [15], casein [12], and hemoglobin [9] and also by hydrolysis of azocasein [12] and insoluble collagen from bovine tendons [16]. Type I collagen was isolated from rat skin [10] and type IV collagen from the bovine