Serum-free organ culture of suckling rat jejunum: Effect of regulatory hormones

Abstract

To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco's modified Eagle's medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h in culture. Dexamethasone increased specific activities of alkaline phosphatase (30%, P < 0.001) and lactase (15%, P < 0.001), and reduced shedding of alkaline phosphatase into the medium (P < 0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P = 0.04) and crypt depth (P = 0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1-3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture.(ABSTRACT TRUNCATED AT 250 WORDS)