ALTERED GROWTH AND ATTACHMENT OF RABBIT CRYPT COLONOCYTES ISOLATED FROM DIFFERENT DEVELOPMENTAL STAGES

The rabbit colon was used to establish an in vitro model for examining development-related cellular changes in colonocyte function, Colonic epithelia from newborn, weanling, and adult animals were separated fro m the muscle and subjected to enzymatic digestion, A mixture of 0.05% Pronase, 0.015% collage-nase IV, and 0.023% DTT was determined to be o ptimal for the isolation of newborn and weanling colonocytes. This sol ution yielded significantly more cells and of greater viability than a 0.1% Pronase, 0.03% collagenase IV, 0.07% DTT mixture that is optimal for adult colonocytes. The epithelial origin of the colonocytes was c onfirmed by immunofluorescent staining of cytokeratins. The isolation procedure resulted in a crypt-enriched population and the cell yield/g of mucosa increased with age as did the crypt depth, Colonocyte viabi lity of adults but not of newborns and weanlings, declined from 24 to 72 h. When grown on plastic, the newborn and weanling colonocytes show a similar to 2-fold increase in number, DNA and protein content over 48 h. In contrast, for all three parameters the adult colonocytes reve aled only a similar to 10% increase, The colonocytes also showed an ag e-related decline in attachment to extracellular matrices. Colonocytes showed maximal attachment to Matrigel and collagen N; newborn and wea nling colonocytes show >80% attachment, whereas adult colonocytes show ed only a 45% attachment. The efficacy of attachment to Matrigel compa red with that on plastic also differed with age, representing 9.3-, 5. 5-, and 4.4-fold increase in adult, weanling, and newborn colonocytes, respectively, New born and weanling colonocytes grown on Matrigel for 48 h, showed a significant, 15% increase in cell number, DNA, and pro tein content compared with those grown on plastic. There was no differ ence in these parameters when adult colonocytes grown on Matrigel were compared with those grown on plastic. In summary, we have established an in vitro model for studying colonic epithelial cells at different stages of development.