Method for Large-Scale Isolation of Pancreatic Islets by Tissue Culture of Fetal Rat Pancreas

Abstract

Detailed studies of the maturation of stimulus-secretion coupling of the pancreatic B-cell requires a supply of isolated fetal islets, which has so far been difficult to obtain. To overcome this problem we have maintained minced and mildly collagenase-digested fetal rat pancreatic glands (21.5 days gestational age) in tissue culture to enable degeneration of the acinar part, leaving the endocrine cells in an isolated and surviving state. Indeed, after 1 wk in culture there was a complete separation between acinar and endocrine cells with the appearance of numerous discrete islets and the disappearance or dedifferentiation of the exocrine cells. Isolated islets were either free floating or attached on top of a monolayer of fibroblast-like cells. Their number after 1 wk in culture was estimated as about 90 per explanted fetal pancreas and a total yield of about 5000 isolated islets was easily achieved. Both light arid electron microscopic examinations showed an excellent structural preservation with a marked predominance of well-granulated B-cells. Numerous islets of the same weight as that measured in cultured islets of adult rats were regularly found after 1 wk in culture. The insulin concentration of the cultured fetal islets was related to the glucose concentration of the growth medium. A similar relationship was found with respect to the insulin release in response to glucose. Thus, fetal islets cultured for 8 days in growth media containing 11.1 or 22.2 mM glucose showed a marked and significant insulin response to glucose in batch-type incubations at the end of the culture period. By contrast, the glucose stimulation of insulin release was insignificant in islets cultured at 5.5 or 2.8 mM glucose. When the culture period was confined to 1 day, there were no effects of glucose on the insulin release irrespective of the glucose concentration of the growth medium. It is concluded that the present technique for tissue culture of fetal rat pancreas makes it possible to isolate substantial amounts of fetal islets predominantly composed of B-cells. The transition in vitro from a poor glucose sensitivity to an adult-type insulin response indicates that the technique can be used in further detailed studies of the molecular mechanisms involved in the growth and development of the pancreatic B-cell.

Phorbol ester stimulation of pancreatic β‐cell replication, polyamine content and insulin secretion

“…The findings presented here confirm that TPA provokes secretion of insulin from the p-cells, and also suggest that the phorboi ester treatment leads to stimulation of p-cell replication. Because islets prepared according to the presently employed technique contain >90% t-cells it seems reasonable to assume that the results are representative of this particular cell type [10]. In addition, incorporation of [aH]thymidine over a 5-h labeling period has been shown to represent p-cell mitotic activity [25].…”

Glibenclamide inhibits islet carnitine palmitoyltransferase 1 activity, leading to PKC-dependent insulin exocytosis

“…In each experiment, all test groups received the same amount of solvent used to dissolve various drugs. We chose to use fetal islets to obtain islet tissue enriched in ␤-cells; previous studies have shown that fetal islets contain 90-95% ␤-cells, which display a full secretory response to glucose after a 5-day culture period similar to that of the adult ␤-cell (20,39).…”

“…Pregnant Wistar rats, purchased from B & K Universal (Sollentuna, Sweden), were killed by cervical dislocation on day 21 of gestation and the fetuses rapidly removed. Islets were prepared from pancreatic glands as previously described (20,39). Briefly, the pancreata were finely chopped and digested for a short time with collagenase.…”

Effect of iodoacetic acid on maintenance of pancreatic endocrine cells of the neonatal rat.

“…Up to now it has been widely acknowledged that culture of fetal and neonatal B cells in media with high glucose is beneficial to their function in vitro. For example, HellerstrOm et al (1979) showed that fetal islets cultured for 8 days in medium containing 11.1-22.2 mM D-glucose responded to an acute glucose stmulation, while there was no response by islets cultured in 5.5 mM D-glucose. Furthermore, in cultured islets the low glucose decreased the cAMP response to D-glucose, which is assumed to be involved in glucose-induced insulin release or B-cell replication (Grill and Cerasi, 1974 ;Ravinovitch et al, 1978 ;Rabinovitch et al, 1980).…”