Development of embryonic chick insulin cells in culture: Beneficial effects of serum-free medium, raised nutrients, and biomatrix

Abstract

A previous finding that insulin cells do not survive or differentiate in explants of embryonic avian pancreas cultured in collagen gel with a serum-containing medium has provided a model system for identification of conditions favorable for development of these cells. To this end, we here modify the substrate and the medium. The epithelial component of dorsal pancreatic buds of 5-d chick embryos was cultured for 7 d on Matrigel in serum-containing and in serum-free medium, the latter incorporating insulin, transferrin, and selenium. Endocrine cell types were distinguished by immunocytochemistry; insulin cell counts were expressed as a proportion of insulin plus glucagon cells. With serum-containing medium, Matrigel stimulated a significant increase in this proportion as compared with collagen gel--3.1% as against 0.2%; the serum-free medium further increased this proportion to 17.3%. Raising the level of essential amino acids approximately fivefold increased the latter figure somewhat (to 18.9%), but it was more than doubled (to 37.4%) by raising the glucose concentration from 10 mM to 20 mM. Raising the levels of amino acids and glucose simultaneously yielded a lesser increase (to 31.8%). Some cultures grown in collagen gel and serum-containing medium for 7 d were transferred to Matrigel and serum-free medium for a further 7 d. Insulin cell development recovered, indicating that progenitor cells had survived and were stimulated to develop by the improved conditions. This study indicates that components of the biomatrix and the medium (in particular, a raised glucose concentration) are important for the survival and differentiation of embryonic insulin cells.

Morphogenesis and differentiation of the avian endocrine pancreas, with particular reference to experimental studies on the chick embryo

“…Replacement of collagen gel with Matrigel, culture conditions otherwise remaining identical, also resulted in a significant increase in the proportion of insulin cells (from 0.2 to 3.1%). This indicates that for survival and development of insulin cells, Matrigel is superior to collagen gel (Rawdon and Andrew, 1997). This finding is in line with results of studies on mammalian islet cells that have shown extracellular matrix produced by cultured bovine corneal endothelial cells (Beattie et al, 1991;Hayek et al, 1989) or by a basement membrane-secreting tumour line (Muschel et al, 1986) to improve islet cell function.…”

“…With a view to identification of factors specifically required for differentiation of insulin cells, an in vitro system utilising the dorsal pancreatic bud of the chick embryo has been established (see Andrew et al, 1994;Rawdon and Andrew, 1997;Rawdon and Andrew, 1988). This model system enables the nature of the substrate and medium to be manipulated.…”

Regulation of Embryonic Chick Insulin Cells: Effect of Retinoic Acid and Insulin-Like Growth Factor 1

“…(Benjamin Rawdon and Ann Andrew are acknowledged as members of our research group who carried out the earlier work referred to.) Raised levels of glucose and essential amino acids in a fully defined synthetic serum-free medium have been shown to increase the proportions of insulin cells (1.9-fold) in the dorsal pancreatic buds of chick embryos cultured on Matrigel [Rawdon and Andrew, 1997]. Chick pancreatic buds cultured on Matrigel in a serum-free medium supplemented with nicotinamide also showed increased proportions of insulin cells (a 2.5-fold increase) [Mngomezulu and Kramer, 2000].…”

“…The proportion of insulin cells that develops in culture has been markedly improved by the use of extracellular matrices and a variety of growth factors or nutrients [Swenne, 1985;Swenne et al, 1987;Otonkoski et al, 1988;Rawdon and Andrew, 1997]. (Benjamin Rawdon and Ann Andrew are acknowledged as members of our research group who carried out the earlier work referred to.)…”

“…When chick pancreatic buds were treated in culture (see Rawdon and Andrew [1997; for culture methods) with all-trans-retinoic acid, the proportion of insulin cells increased in a doserelated manner; a concentration of 10 -6 M resulted in a twofold increase in the proportion of insulin cells in comparison to a higher dose of RA (10 -5 M). However, the increase in the proportion of insulin cells was 3 times that of insulin cells in control explants (F12.ITS or F12.ITS + DMSO) [Penny and Kramer, 2000].…”