Isolation and characterization of attached and nonattached populations of rat colon epithelial cells in culture

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 hours of Incubation. The yield of cells averaged about 153×10⁶/rat, with a standard deviation of 12.9×10⁶. 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 hours 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]thymldine 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]urldine into RNA was distinctly higher in the attached cell population when compared to the nonattached population, although both populations of cells actively Incorporated [¹⁴C]glyclne into protein. Whereas total colon cells appeared heterogeneous In cytomorphology, the attached cells were more uniform and smaller and contained deeply hematoxylinophilic, round nuclei. With the use of short-term tissue culture techniques, we have separated a subpopulation of colon cells exhibiting characteristics compatible with the less differentiated Immature cryptal cells.