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 Ds-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 13 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 45Ca could also be measured in everted duodenal sacs, subsequent to culture under similar conditions, and was also increased by vitamin D3 in the medium . Other embryonic organs, esophagus, stomach, liver, pancreas, lung, skin, and muscle, did not produce CaBP in response to vitamin D3 in the culture medium . However, CaBPsynthesizing capacity was present in the entire intestinal tract, exclusive of the rectum . 59Fe and 82P uptake by cultured duodenum were also stimulated by vitamin D3 . 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 .

Decrease in serum immunoreactive parathyroid hormone in rats and in parathyroid hormone secretion in vitro by 1,25-dihydroxycholecalciferol.

“…1,25-(OH)2Ds at 10 nM also stimulates calcium-45 uptake by intestine in vitro (35). Thus, apart from the uncertainty of the issue of free 1,25-(OH)2D3 concentration, it would seem that the concentration of 1,25-(OHi)2D3 we used in our in vitro work is probably in the physiological range.…”

Vitamin D stimulates (Na+ + K+)‐ATPase activity in chick small intestine

“…Methods used for organ culture of embryonic chick small intestine and for determination of Rb+ and Ca2+ uptake by cultured guts have been detailed in [5, 6,9]. Briefly, intestinal segments excised from chick embryos between day 15-20 were cultured (for 12-48 h) in serum-free McCoy's 5A mod.…”

Maintenance of embryonic chick duodenum in large scale organ culture

“…There is every reason to expect that the procedure would be adaptable to the study of factors affecting intestinal cell structure and/or a variety of intestinal functions. The system is one for maintenance of duodenal structure and function, however, since cell proliferation is reduced (3).…”

“…Total protein, total DNA and radiolabeled DNA and protein synthesis have been monitored in the cultured duodenum, as a function of treatment, by previously published techniques (3,4).…”