Studies of the morphology and endocrine function of in vitro cultures of neonatal hamster pancreatic tissue

• Main Author: Beeson, Virginia S.
• Language: en_US
• Published: Boston University 2019
• Online Access: https://hdl.handle.net/2144/34448

Thesis (M.A.)--Boston University === PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [e-mail]. Thank you. === The present research was designed to investigate the optimal in vitro conditions requisite for the successful growth of pancreatic endocrine tissue in monolayer cultures and to determine the morphology and endocrine functional characteristics of such cultures. Pancreatic tissue obtained from neonatal (24 hrs to 5 days old) golden hamsters, Hesocricetus auratus, v-ras cultured on coverslips and in milk dilution bottles using Eagle's basal medium supplemented with 20% inactivated calf serum. Several other donor tissue sources and types of culture media also Here explored. The medium from a successful culture lias removed at 1 to four day intervals and stored at -15° C for analysis of insulin-like material. Coverslip cultures were analyzed histochemically using aldehyde fuchsin and Harris hematoxylin and using hematoxylin and eosin stains. The culture medium was extracted by the Scott-Fisher method and the protein extracts partially purified by filtration on Sephadex G75. The insulin-like material present in these extracts was assayed by standard methods using the rat epididymal fat pad and by radioimmunoassay using the double antibody technique. Preliminary studies were performed to determine the optimal tissue donor source and culture conditions for cell growth. The most successful cultures were derived from neonatal hamster pancreata (24 hrs to 5 days old) cultured in E~gle's basal medium supplemented with 20% inactivated calf serum. However, donor tissue obtained from the adult hamster, mouse islet lesions, or hamster pancreatic homografts were not cultured successfully in the Eagle's basal medium supplemented with 10 or 20% inactivated calf serum. Furthermore, adult or neonatal hamster pancreatic tissue did not grow in lactalbumin hydrolysate medium. A single culture o:f cells derived from pancreatic tissue of five day neonatal hamsters was grown for over thirty days. The culture successfully underwent two cell passages at 10 day intervals and contained viable cells during this time. The cells in coverslip culture were of the fibroblastic and epithelioid types and most contained granules which were coarser for the former than for the latter cell type. Those of the epithelioid, but not the fibroblastic, cells were stained specifically by aldehyde fuchsin in cultures ten and twenty-two days of age. The presence of these specifically staining granules and other morphologic characteristics suggest that the epithelioid cells may be identical with the beta cells of the islets of Langerhans. The bioassay data indicated that a material was released into the medium by the cells in culture which stimulated the uptake of glucose by rat adipose tissue and which was capable, furthermore, of reacting with guinea pig antibodies to human insulin. Nonincubated culture medium and medium in which non-pancreatic cell lines were incubated gave negative results. It may be concluded that the epithelioid cells grown in culture boar a morphological and possibly a functional resemblance to beta cells of the islets of Langerhans. === 2031-01-01