LARGE-SCALE PRODUCTION OF FETAL PORCINE PANCREATIC ISLETLIKE CELL CLUSTERS

Abstract

A recently described method for the preparation of isletlike cell clusters (ICC) from human fetal pancreas has been applied to the fetal pig with the ultimate aim of large-scale production of ICC. Fetuses ranging in age from 51 to 77 days were used, and after a brief colla-genase-incubation the pancreatic digest was plated into culture dishes containing medium RPMI 1640 supplemented with either 10% fetal calf serum (PCS) or human serum (HS). HS seemed to increase the number of ICC formed as compared to that obtained with FCS. A total of more than 100,000 ICC were produced from each of 3 litters, ages 67–77 days, after culture in the presence of HS. The DNA content of such ICC was reduced by about 50% as compared to those maintained with FCS supplementation. Immunocytochemical staining revealed insulin-and glucagon-positive cells scattered among a majority of nonstained cells within the cell clusters. ICC maintained in either FCS or HS displayed significant rates of (pro)insulin biosynthesis in vitro and an increased insulin release when exposed to 16.7 mM glucose plus 5 mM theophylline. Four weeks after implantation, ICC grafted under the kidney capsule of nondiabetic nude mice contained frequent insulin-and glucagon-positive cells. In 2 nude mice transplanted with ICC, the functional capacity of the graft was tested by perfusing the graft-bearing kidney. When the perfusion fluid was changed from one containing 2.8 mM glucose to one containing 16.7 mM glucose ± 5mM theophylline, the secretion of insulin increased within a few min. It is concluded that the fetal porcine pancreas can be used for large-scale production of ICC, which have a very consistent, but immature functional capacity. Because of their inherent growth and differentiation properties, fetal porcine ICC constitute a potential source of xenogenic islet grafts intended for human diabetics.