Pancreatic beta cell culture: preparation of purified monolayers

Procedures were developed for preparing partially purified beta cell monolayer cultures. Neonatal rat pancreases were dissociated with a trypsin-collagenase solution. Beta cells were separated from denser acinar cells by centrifuging the initial suspension in a two layer discontinous Ficoll gradient (20, 25%). Resultant cultures, highly enriched in beta cells, were further purified by incubation with cystine-free medium. This caused necrosis of the majority of fibroblastoid cells within two days, while beta cells were considerably less affected. The resultant cultures contained an average of 72% beta cells compared to 10% in untreated control cultures. Insulin release by purified monolayers remained responsive to changes in the glucose concentration of the culture medium.