Preparation of single-cell suspensions from normal liver, regenerating liver, and Morris hepatomas 9121 and 5123tc.

Abstract

Suspensions of single cells were prepared from normal adult rat liver either by mincing the tissue and incubating it in various concentrations of sodium tetraphenylborate, ethylene-diaminetetraacetate, and 0.1% hyaluronidase plus 0.05% collagenase or by continuous recirculating perfusion of the liver with 0.1% hyaluronidase plus 0.05% collagenase. Only the latter method produced cells in good yield that were not stained with trypan blue. Incorporation of l-leucine-1-14C into protein was up to 60 times higher in cells prepared by continuous perfusion with hyaluronidase plus collagenase than in cells prepared by incubation of the minced tissue in sodium tetraphenylborate or in hyaluronidase plus collagenase. Liver cells prepared by continuous perfusion had a better preserved ultrastructure than cells obtained by the other methods. In regenerating liver, cells that were not stained with trypan blue were obtained by the two methods with hyaluronidase plus collagenase, the yield being highest after continuous perfusion. In Morris hepatomas 9121 and 5123tc, continuous perfusion could not be established. However, mincing the tissue and incubating it in hyaluronidase plus collagenase yielded cells with an intact ultrastructure. Those cells excluded trypan blue. The amount of the incorporation of l-leucine-1-14C into protein was about 300 times higher than in cells prepared by sodium tetraphenylborate.