Monolayer Culture of Pancreatic Islet Cells of the Adult Rat : Effect of 2-Deoxy-2-Fluoroglucose

Abstract

In the present study, the culture system for preparing monolayer islet cells of the neonatal rat was applied and modified for use with the pancreas of the adult rat. In this procedure, whole pancreatic tissues were enzymatically dispersed and then cultured for 30 days in TCM 199 medium with either 5.5 mM glucose or 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose. Under culture conditions without 2-deoxy-2-fluoroglucose, the responsiveness of B cells was totally abolished by day 20 of culture. The addition of 2-deoxy-2-fluoroglucose destroyed fibroblasts selectively in a period of 20 days, yielding the monolayers mostly consisting of islet cells, and the morphological characteristics were well preserved at the end of the culture study period. After culture for 20 days in medium with 2-deoxy-2-fluoroglucose, insulin secretion was raised in a dose-dependent fashion due to the increasing concentrations of glucose, leucine and 2-ketoisocaproate. The dose-response curve for insulin secretion evoked by glucose was sigmoid with a Km of 7 mM glucose, and the secretion threshold was observed at a concentration of between 2.8 and 5.5 mM glucose. The ratios of the maximum level to the basal were 6, 5 and 3 respectively for glucose, leucine and 2-ketoisocaproate. The secretory competence was preserved in the B cells on day 30 as well. Addition of epinephrine or clonidine inhibited the glucose-induced insulin secretion dose-dependently. At a concentration of 10(-7) M, both drugs produced an 80% drop in insulin secretion evoked by glucose. The inhibitory effect of epinephrine or clonidine was reversed by 3 X 10(-5) M yohimbine or 10(-5) M phentolamine, whereas 10(-5) M propranolol had little or no effect and alpha adrenergic blockade of prazosin (5 X 10(-5) M) was weak as compared to that of yohimbine. At a high concentration (10(-5) M) of phenylephrine, a marked drop of insulin secretion was observed. In summary, the present culture system facilitates the establishment of monolayers of adult rat pancreas that consist mostly of islet cells. In addition, it is certain that the response of the B cells in 2-deoxy-2-fluoroglucose to nutrient secretagogues and the adrenergic modulation of insulin secretion are well preserved for a long-term culture period of 30 days. These preparations may provide a useful tool not only for the in vitro study of the B-cell function, but also for use in implantation resource.

Selection of polymers for application in scaffolds applicable for human pancreatic islet transplantation

“…To exclude the fibroblasts from the proliferating cells, islets were cultured in the presence of 2-deoxyglucose ( figure 4(B)). The 2-deoxyglucose selectively kills fibroblasts [29,30]. The expression of the EMT markers decreased approximately 50% in the 2-deoxyglucose after 21 d in cultures.…”

“…In one of the series of experiments 5 mM 2-deoxyglucose (Sigma-Aldrich) was added to the cultures. The 2-deoxyglucose is selectively toxic for fibroblasts and was used to delete fibroblasts from the culture [29,30]. stop solution using a microplate reader (VersaMax; Molecular Devices).…”

“…In one of the series of experiments 5 mM 2-deoxyglucose (Sigma-Aldrich) was added to the cultures. The 2-deoxyglucose is selectively toxic for fibroblasts and was used to delete fibroblasts from the culture [29,30] stop solution using a microplate reader (VersaMax; Molecular Devices). Normal islet function was defined as at least a two-fold increase in insulin levels in the high glucose incubation compared to the first low glucose incubation.…”