Variations in Cell Overlapping and Growth Depending on the Substratum

Abstract

The inhibition of growth under conditions of cell crowding has been studied in cultures of several types of normal and virus transformed cells in many laboratories (1). In some cases the data suggested that the transformed lines had a decreased sensitivity to the inhibition of division but that the ultimate expression of the phenomenon depended on the affinity of the cells with the substratum (2, 3). To verify this hypothesis one would need a surface for the growth of cell monolayers where cell attachment could be easily modified. The present work describes the growth of Earle's L mouse cell line (4) on such a surface made of a film of a water insoluble protein polymer (5) which could have broader applications in tissue culture.

Materials and Methods. Bovine serum albumin (BSA) was polymerized with glutaraldehyde by adding with constant stirring 1.2 ml of a 2.5% aqueous solution of glutaraldehyde to 1 ml of a sterile 30% solution of BSA in 0.1 M phosphate buffer pH 6.8 (5). A volume of this solution adequate to make a layer 2—3 mm thick was quickly poured on the surface of 30 ml plastic Falcon bottles and the protein was allowed to polymerize for 3 hr at room temperature. Following polymerization the protein was covered for 24 hr with borate buffer 0.1 M pH 8.4 or with borate buffer containing 1 mg/ml of either polyglutamic acid (molecular weight 45,000), polylysine (molecular weight 175,000) or calf thymus histones. The polymer was then washed twice with culture medium and the cells were seeded. Control cultures were grown on noncoated plastic surfaces. The cells were maintained in Eagle's minimal essential medium supplemented with 10% calf serum and 50 μg/ml aureomycin.