Isolation and characterization of attached and nonattached populations of rat colon epithelial cells in culture
Mazumder, A.; Patnaik, R.N.; Bhagavan, B.S.; Nair, P.P.
Journal of the National Cancer Institute 62(6): 1443-1449
1979
ISSN/ISBN: 0027-8874 PMID: 286117 Document Number: 143097
Colon epithelial cells of Sprague-Dawley rats were isolated by the incubation of everted colon sacs aerobically in Puck's Saline F containing 0.5% hyaluronidase and a mixture of antibiotics (penicillin, streptomycin and Fungizone). The complete removal of both mature absorptive and deep cryptal proliferating cells required about 2 h of incubation. The yield of 12.9 .times. 108. Approximately 98% of these cells excluded trypan blue and were capable of sustained oxidation of [14C]glucose. When these cells were placed in short-term culture for about 16 h. In Eagle's minimum essential medium containing 15% calf serum and antibiotics (penicillin, streptomycin and Fungizone), about 15 .+-. 2% attached to the surface of the culture plate. Attached cells showed a 15-fold higher incorporation of [3H]thymidine into DNA than did nonattached cells . Thymidine kinase activity, generally associated with rapid growth and increased DNA synthesis, was mostly localized in the attached cells. Incorporation of [3H]uridine into RNA was distinctly higher in the attached cell population when compared to the nonattached population, although both populations of cells actively incorporated [14C]glycine into protein. Whereas total colon cells appeared heterogeneous in cytomorphology, the attached cells were more uniform and smaller and contained deeply hematoxylionophilic, round nuclei. With the use of short-term tissue culture techniques, a subpopulation of colon cells exhibiting characteristics compatible with the less differentiated immature cryptal cells was separated.