Multiple hormone requirement for the synthesis of alpha 2u-globulin by monolayers of rat hepatocytes in long term primary culture.

Primary cultures of adult rat hepatocytes were prepared by releasing liver cells through fractional trypsinization and by a novel nonenzymatic procedure involving the imprinting of liver explants on the surface of the culture flask (explant imprinting). The comparative properties of these two primary cultures and their abilities to synthesize alpha 2u-globulin under multihormonal stimulation were investigated. The hepatocytes isolated from male rats by fractional trypsinization were found to divide rapidly and form a confluent monolayer within 5-7 days of culture. However, these cells failed to synthesize alpha 2u-globulin even after treatment with 5 alpha-dihydrotestosterone, GH, T3, and dexamethasone, hormones with known stimulatory effects on the synthesis of alpha 2u-globulin in vivo. On the contrary, hepatocytes prepared from male rats by the nonenzymatic procedure of explant imprinting were found to synthesize alpha 2u-globulin for more than 2 weeks and responded to GH, T3, and dexamethasone with enhanced production of this protein. Fortification of the culture medium with all of the above three hormones caused an approximately 9-fold enhancement of alpha 2u production within 4 days of culture compared to production in control culture without these hormones. The above results substantiate earlier findings concerning the multiple hormonal requirement for hepatic synthesis of alpha 2u-globulin in vivo and show that isolation and culture of hepatocytes by nonenzymatic explant imprinting may serve as a useful procedure for studying endocrine regulation of gene activity in vitro.