Monolayer Cell Culture of Neonatal Rat Pancreas: Light Microscopy and Evidence for Immunoreactive Insulin Synthesis and Release

Abstract

Pancreatic cells from neonatal rats, isolated by repeated exposure to a mixture of trypsin and collagenase, were cultured in monolayer for periods up to 19.5 days. This preparation was characterized by: (1) light microscopy combined with morphometric analysis; and (2) measurements of immunoreactive insulin (IRI) and protein content of cultured cells and of the amounts of IRI released into the culture medium. The major problem encountered was the rapid proliferation of fibroblastoid cells which tended to overgrow the culture. The decantation of the primary cell suspension after 14 hrs of culture produced a preparation enriched in epithelioid cells, which appeared as clusters. Contained in these clusters were aldehyde-thionin positive cells, presumably B cells. The number of clusters of epithelioid cells did not change over an 8.5 day culture period, but the size of the individual clusters diminished with time. The protein content of cultured cells, which probably reflects the degree of contamination by fibroblastoid cells, increased progressively up to 8.5 days of culture. IRI content tended to increase as culture time progressed, despite the relatively large amounts of IRI which were released into the culture medium during that period. Pancreatic cells cultured for periods of 3.5 and 5.5 days were used to study the effect of glucose alone or together with theophylline on IRI release during a 4 hr incubation period in a Krebs-Ringer bicarbonate buffer. Increasing glucose concentration from 5.5 to 11 HIM markedly stimulated IRI release. The presence of theophylline enhanced the effect of glucose at both low and high concentrations. These results indicate that B cells can be grown in monolayer culture and maintain their capacity to synthesize IRI and to release it under specific stimulatory conditions. (Endocrinology90: 239, 1972)