Monolayer Culture of Adult Rat Pancreatic Islets on Extracellular Matrix: Long Term Maintenance of Differentiated B-Cell Function*

Abstract

Fragmented islets, obtained by mild overdigestion of the adult rat pancreas with collagenase, readily formed monolayer cultures on dishes coated with extracellular matrix derived from bovine corneal endothelial cells. Contaminating fibroblasts were removed by treatment with sodium ethylmercurithiosalicylate. The cultured islets remained functional for over 6 weeks in primary culture and up to 9 weeks in secondary culture, as indicated by their substantial insulin response to an acute glucose stimulus. Insulin secretion from islet monolayers showed biphasic kinetics. The functional competence of the monolayers was further evaluated by studying glucose-stimulated insulin release in the presence of various modulators of B-cell function. The response to physiological agents such as somatostatin, epinephrine, glucagon, and arginine was retained for at least 4 weeks in culture. The sensitivity to inhibition by somatostatin and epinephrine (ID50 = 10 ng/ml) and that to stimulation by glucagon (ED50 = 3 ng/ml) were similar to or better than those for freshly isolated islets. We have thus obtained a fibroblast-free monolayer culture of pancreatic islets from adult rats containing B-cells that retain normal function for long periods. This experimental system appears ideally suited for studying chronic modulations of islet cell function under controlled in vitro conditions, which can allow the stimulation of normal and diabetic environments.