A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 ,ug of insulin per mg of cell protein.[3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4 polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-l-methylxanthine. Insulin secretion at ogtimal glucose concentration (7.5 mM) was 2.4 milliunits per 10 cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.Much of the current understanding of insulin biosynthesis and beta cell metabolism has been derived from in vitro studies utilizing isolated islets, either intact or dissociated in monolayer culture. Such studies have been limited by (i) the difficulty of preparing even small quantities of islets, (ii) cellular and hormonal heterogeneity within islets, and (iii) rapid loss of insulin production in vitro. The recent availability of a highly differentiated rat insulinoma tumor line (1) has provided experimental material for biochemical studies including purification and characterization ofpreproinsulin mRNA (2) and cloning and sequence analysis ofcDNA (3). Further in vitro studies would be facilitated by the availability of permanent cell lines possessing functions characteristic of differentiated beta cells. Attempts to establish stable insulin-producing cell lines from primary islet monolayer cultures (4-6), insulinomas (7,8), and hybrids ofislet cells and continuous cell lines (9) have met with only limited success. Simian virus 40 (SV40) transformation of rat islets has yielded continuous cell lines that produced a 30,000-dalton protein antigenically related to insulin (10). Rae et al. (11) have recently derived continuous cell lines from the poorly differentiated Kirkman hamster insulinoma which did not make insulin in vitro but resumed insulin synthesis in vivo.In this communication we describe a continuous beta cell line, established from SV40-transformed Syrian hamster islet cells, that produces substantial quantities of insulin and proinsulin in vitro. Preliminary studies characterizing the insulin product and comparing stimulus-secretion coupling between this transformed hamster beta cell line (HIT) and primary islet cultures are also described.MATERIALS AND METHODS Isolation of Pancreatic Islets. Pancreata were dissociated by the collagenase-digestion technique of Lacy and Kostianovsky (12). DNase I (Worthington; code DP, 23 units/ml) was added ...

No abstract

No abstract

The mode of sterilization (filtration or heat) was found to significantly affect the activity of trypsin solutions. Trypsin activity was substantially reduced in the initial fractions of filtrate passed through asbestos filter pads; heat-sterilized trypsin was satisfactory for transfer of cell cultures grown on glass. Heat-sterilized trypsin may be useful when elimination of filterable organisms is required.

The mode of sterilization (filtration or heat) was found to significantly affect the activity of trypsin solutions. Trypsin activity was substantially reduced in the initial fractions of filtrate passed through asbestos filter pads; heat-sterilized trypsin was satisfactory for transfer of cell cultures grown on glass. Heat-sterilized trypsin may be useful when elimination of filterable organisms is required.