Tissue Culture of Fetal Rat Islets: Comparison of Serum-Supplemented and Serum-Free, Defined Medium on the Maintenance, Growth, and Differentiation of A, B, and D Cells*

Abstract

Fetal rat pancreatic islets were grown in tissue culture in medium RPMI 1640 supplemented with 10% heatinactivated fetal bovine serum or with a mixture of nine hormones and growth factors. Modifications of the medium and the culture surface were made in an attempt to promote attachment of the islet tissue when plated in the serum-free medium. The maintenance of the islet cells was assessed throughout 30 days in culture by RIAs of insulin, glucagon, and somatostatin in extracts of the cultured tissue. The changes in the total number of cells and in the numbers of B, A, and D cells in vitro were estimated by counts of the hormone-positive cells after immunocytochemical staining of cytocentrifuged cell smears. The responsiveness of the B cell to glucose-stimulated insulin release was determined weekly during the culture period. The insulin content and B cell number increased during the first week in culture in both defined and undefined media. By the criteria measured, the serum-free medium preserved the B cells to the same degree as the serum-supplemented medium. B cell secretory response to glucose was also maintained in the absence of serum. A cells were not maintained in either medium. The number of A cells decreased to barely detectable levels by 15 days in culture. The D cells were not maintained in the serum-supplemented medium, but in the serum-free medium, the D cells and somatostatin content of the tissue were preserved at higher levels for a longer period in vitro. The pancreatic tissue could not be plated in the serum-free medium, since the islet cells failed to attach to the culture surface. The data indicate that fetal rat islet cells differ in their responses to the in vitro environment. In addition, the factors included in the serum-free medium can, after attachment, support all three of the islet cells examined at least as well as serum, and in the case of the D cells, to a greater extent. The formulation of this defined medium will provide a means to examine empirically the effects of nutrients and other defined growth and tissue factors on the maintenance, growth, and differentiation of these important endocrine cells under controlled conditions in vitro.