EMBRYONIC CHICK INTESTINE IN ORGAN CULTURE

Abstract

Duodena from 20-day-old chick embryos can be maintained in large scale organ culture on specially designed stainless-steel grids in contact with serum-free medium for 48 h with excellent preservation of mucosal structure at both the light and electron microscope levels. Although mitotic rate was subnormal, several other factors attest to the essential viability of the cultured intestine: L-leucine incorporation into protein, as well as the synthesis of a specific vitamin D(3)-induced calcium-binding protein (CaBP), increased over a 48-h culture period, and the electropotential gradient across the intestine was maintained throughout the culture period as was a concentration gradient for calcium. The tissue responded to vitamin D(3) in the medium by synthesizing the calcium-binding protein within 6 h and by exhibiting enhanced (45)Ca uptake within 12-24 h. Concentrations of vitamin D(3), or its 25-hydroxylated derivative, higher than necessary for CaBP induction, also increased the activity of alkaline phosphatase. The 1,25-dihydroxylated derivative of vitamin D(3), at a level extremely potent in CaBP induction, did not stimulate alkaline phosphatase. Mucosal to serosal transport of (45)Ca could also be measured in everted duodenal sacs, subsequent to culture under similar conditions, and was also increased by vitamin D(3) in the medium. Other embryonic organs, esophagus, stomach, liver, pancreas, lung, skin, and muscle, did not produce CaBP in response to vitamin D(3) in the culture medium. However, CaBP-synthesizing capacity was present in the entire intestinal tract, exclusive of the rectum. (59)Fe and (32)P uptake by cultured duodenum were also stimulated by vitamin D(3). The system has proven quite useful in the study of the vitamin D-mediated calcium absorptive mechanism but should be applicable to the study of the absorption of other nutrients, drugs, hormones, etc., as well as other studies of intestinal function.