Hypoosmotic Exposure of Canine Pancreatic Digest as a Means to Purify Islet Tissue

Abstract

The development of more effective means to separate pancreatic islets from the unwanted exocrine tissue would greatly advance the field of clinical islet allotransplantation in the treatment of insulin-dependent diabetes mellitus. Recent experiments with hamster islets have demonstrated a selective destruction of dissociated single exocrine cells when exposed to hypotonic conditions. It was the aim of this study to extend these observations to the canine model with collagenase dissociated pancreatic tissue and to evaluate the treatment's effect on islet function. Pancreases from five mongrel dogs were digested using an automated protocol of intraductal delivery of collagenase, and gentle dissociation. Duplicate samples of pancreatic digest were removed for insulin and amylase determination prior to and immediately following exposure to 50 mOsm/kg salt solution for a period of 30, 60, or 300 s before returning the digest to isoosmotic conditions. The remaining digest was cultured for a period of 48 h at 37°C before the tissue was recombined, washed, and a third sample removed for insulin and amylase. In vitro viability was then assessed using a static incubation assay with insulin content measured using a double-antibody radioimmunoassay, and amylase was determined using a colorimetric assay system. No difference in the insulin or amylase levels between the experimental groups was observed immediately following the hypotonic exposure; however, a significant decrease in the amylase content was observed following the 48-h culture period in digest that had been hypoosmotically exposed for 60 or 300 s compared with the pretreatment group (2.83 ± 0.41 IU amylase/mg pancreas vs. 1.29 ± 0.21 and 0.83 ± 0.12, mean ± SEM, p < 0.05). Insulin content was also significantly reduced in the 300-s exposure group compared with nontreated controls (3.2 ± 0.6 mU insulin/mg pancreas vs. 2.0 ± 0.2). The insulin/amylase ratio (I/A), a measure of islet and exocrine content, was 1.1 ± 0.13 following pancreas dissociation and 1.34 ± 0.21 for control tissue cultured for 48 h. The I/A ratio increased following hypoosmotic exposure to 1.50 ± 0.31 for tissue exposed for 30 s, 1.77 ± 0.19 for 60-s exposure, and 2.54 ± 0.13 for tissue exposed for 300 s (p < 0.05, vs. pretreatment group). In vitro insulin secretion was equivalent with the exception of the tissue exposed for 300 s, which had an increased basal level of insulin resulting in a significantly decreased stimulation index (3.8 ± 0.5 vs. 8.1 ± 1.2 for the purified islet control group, p < 0.05). These results suggest that a brief hypotonic exposure to pancreatic digest can alter the insulin/amylase ratio; however, there is a functional impairment on subsequent islet function after a period of in vitro tissue culture.