Enzymatic and mechanical requirements for the dissociation of cortical cells from rat adrenal glands

Abstract

Cell yield and steroid synthetic responses (to no addition, to ACTH, 3′,5′-AMP, or NADPH) were determined in suspensions of rat adrenocortical cells following systematic manipulation of the conditions of cell dissociation. Preincubation in 0.25% trypsin compared to medium alone, followed by dissociation in a mixture of collagenase and hyaluronidase, improved cell yield and steroid synthetic response. When concentrations of collagenase and hyaluronidase in the dissociation medium were varied, cell yield was found to vary directly as a function of collagenase concentration ( r = 0.938, P < 0.005) but was unrelated to hyaluronidase concentration. The steroidogenic response per cell to both 3′,5′-AMP and NADPH stimulation increased with increasing collagenase concentration up to 0.02%. At higher collagenase concentrations, the NADPH response per cell continued to increase while the 3′,5′-AMP response per cell decreased. Hyaluronidase concentration had a minimal systematic effect on steroidogenic response. Systematic manipulation of time in histolytic enzyme and intensity of mechanical agitation also appeared to alter cell suspension characteristics.