Tissue Culture of Human Fetal Pancreas: Development and Function of B-Cells In Vitro and Transplantation of Explants to Nude Mice

Abstract

The present study evaluates the development and function of human fetal B-cells in vitro with a view to using such cells in future attempts for transplantation of human fetal pancreas to diabetic patients. A method previously described in our laboratory for preparing islets in vitro from the fetal rat pancreas has been applied and modified for use with human fetal pancreas. Pancreatic glands of different gestational ages were obtained from 37 consecutive prostaglandin-induced abortions. After a mild collagenase treatment, the partially disintegrated tissue was maintained in culture for 7 days in tissue culture medium RPM1 1640 plus 20% fetal calf serum to permit cell attachment and outgrowth of endocrine cells. In 17 of the 37 consecutively cultured fetal pancreatic glands, islet-like cell clusters were formed. The 20 remaining glands were lost because of either bacterial contamination or lack of viability already before dissection had occurred. Sections of the newly formed cell clusters revealed well-preserved pancreatic cells showing frequent mitotic figures. The tissue exhibited a high rate of (pro)insulin biosynthesis and a modest insulin response to secretory stimuli, suggesting that the mechanism of glucose regulation by the fetal B-cells is not yet fully developed. Electron micrographs showed a large number of granule-containing cells, some of which were identified as B-cells. In nine cases, harvested cell clusters were implanted beneath the kidney capsule of nude mice. When these animals were killed after 2 mo, seven mice showed a considerable growth of the grafts with numerous islet-like structures containing insulin- and glucagon-positive cells. A high replicatory activity of the grafted cells was further supported by the finding of numerous labeled nuclei in autoradiographs of the graft-bearing kidneys. Since no immunocytochemical staining was carried out, the cell replicatory rate is representative for the whole grafted cell population rather than for the endocrine cells. It isconcluded that this improved culture technique provides a suitable system for the study of growthand differentiation of human fetal pancreas and may facilitate the preservation of pancreatic B-cells intended for transplantation.