A Simple Method for the Preparation of ACTH Responsive Isolated Cells from Rat Adrenals

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A simple and reproducible method for the preparation of responsive rat adrenal cells was developed, using bovine serum albumin (BSA) to prevent the cell rupture occurring in a protein free medium and to buffer the proteolytic action of trypsin. Rat adrenal slices were placed in Eagles minimum essential medium (Eagles MEM) containing 0.25% trypsin and 0.3% BSA at 35° under a 95% O2: 5% CO2 atmosphere and were dispersed by mechanical stirring. After four 15-min digestive incubations, the dissociated cells of the 2nd, 3rd, and 4th incubates were pooled and washed by resuspending in 0.05% soybean trypsin inhibitor-2% BSA in Eagles MEM. The isolated adrenal cells were used by suspending them in 0.2% soybean trypsin inhibitor-2% BSA in Eagles MEM at a concentration of 2×105 cells per ml. Corticosterone production by the isolated cells was induced by porcine ACTH, Gly1-(1-18) ACTH amide, and dibutyryl cyclic 3, 5-adenosine monophosphate. A linear relationship was obtained between the corticosterone production and the log concentration of ACTH over a range of 60μU/ml to 500μU/ml with a precision indexλ of 0.06. Prolonged pretreatment of rats with ACTHZ elicited both an enhanced responsiveness of the isolated adrenal cells and an increased recovery of the responsive adrenal cells. Adrenal slices to be used could be stored overnight at 4°.
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