Purification of dispersed rat adrenal cells by column filtration

Publisher Website
Google Scholar
Abstract
A simple and rapid method for the purification of specific cell populations in dispersed rat adrenal capsular and decapsulated preparations is described. Dispersed cell preparations were filtered by passage through a column of Sephadex G-10 (for decapsulated preparations) or G-15 (for capsular preparations). The smaller cells, i.e., zona reticularis in decapsulated preparations and zona glomerulosa in capsular preparations, appeared in the filtrate and were separated from the larger zona fasciculata cells, which were retained on the column in both situations. In 6 capsular preparations, contamination by zona fasciculata cells was reduced from 7.3 .+-. 1.1% (mean .+-. SE) to 0.28 .+-. 0.09%. The recovery of zona glomerulosa cells was 18.6 .+-. 2.2% of those applied to the column. Corticosterone [B] output per million cells was reduced, after filtration, to 48% of the output by unpurified cells and aldosterone output was reduced to 10-20% of that of unpurified cells. When expressed as a stimulation ratio (S = stimulated output/basal output), the response of B output to 8.4 mM [K+] was not altered significantly after filtration. S values were 3.04 .+-. 0.26 and 2.27 .+-. 0.48 for unpurified and filtered cells, respectively, but the S value for ACTH, which preferentially stimulated the contaminating zona fasciculata cells, decreased significantly from 6.40 .+-. 0.49 to 2.54 .+-. 0.44 (P < 0.001). Both the steroid outputs and S values were similar to those for zona glomerulosa cells after purification by unit gravity sedimentation. Cells retained by the column and retrieved by subsequent resuspension and filtration to remove Sephadex particles showed steroidogenic characteristics similar to those of unpurified cells. Filtration of decapsulated preparations resulted in recoveries of 44.6 .+-. 3.0% of loaded reticularis cells and 4.7 .+-. 0.7% of loaded fasciculata cells after filtering 18 adrenal equivalents for 10 min (28 experiments). In 4 experiments with 36 adrenal equivalents filtered for 5 min, recoveries were 35.9 .+-. 5.1% and 1.5 .+-. 0.4%, respectively. The retained cell preparations showed 50% enrichment of zona fasciculata cells. After filtration according to the former protocol, cells secreted more deoxycorticosterone (DOC) and less B per million cells than either the unpurified or retained cell preparations. The filtered cells were less responsive to stimulation by ACTH, the effect being most marked for B output, for which the S value decreased from 126 .+-. 13 for unpurified and 131 .+-. 4.5 for retained cells to 15.8 .+-. 5.2 for filtered cells. Corresponding S values for DOC were 13.9 .+-. 1.3, 22.7 .+-. 4.7 and 8.3 .+-. 3.0. Basal R (DOC output/B output) values of 0.41 .+-. 0.05, 0.25 .+-. 0.07 and 1.20 .+-. 0.32 for unpurified, retained and filtered cells, respectively, were observed. Steroid outputs and S and R values for filtered and retained cells were, therefore, similar to those for the equivalent zona reticularis and zona fasciculata preparations, obtained by unity gravity sedimentation. The major advantage of the column filtration procedure for preparation of specific cell populations is that it is rapid and simple both in terms of equipment and manpower, while maintaining a high degree of enrichment and acceptable cell recovery levels and yielding cells that are found by both morphological and functional criteria, to be viable.