Experimental islet isolation in porcine pancreas with new enzyme Liberase PI

doi:10.1016/j.transproceed.2004.08.010

Transplantation Proceedings

Volume 36, Issue 7, September 2004, Pages 2197-2199

https://doi.org/10.1016/j.transproceed.2004.08.010 Get rights and content

Abstract

The aim of this study was to investigate the results of 20 consecutive porcine islet isolations using a new enzyme Liberase PI. Twenty pancreata were procured for islet isolation, which was performed using modified Ricordi's method with Liberase PI. Quantitation of islet viability staining, insulin stimulation assay, intracellular insulin content/DNA, and in vivo transplantability into diabetic nude mice were examined for quality control. The results were compared between a high-yield group (>2500 IEQ/g pancreas) and a low-yield group (<2500 IEQ/g pancreas). Sufficient amount of purified islets (3000 IEQ/g pancreas) were obtained using the new brand enzyme Liberase PI. These islets showed good quality in structure and functions, which were demonstrated by in vitro and in vivo standard assays. Isolation index (IEQ/number) of the low-yield group was lower than that of high-yield group (0.75 vs 0.86), which means more fragmentation of islets in the low-yield group. There were no differences in function between the two groups. In conclusion, we obtained sufficient numbers of viable, functional islets from porcine pancreas using a new brand enzyme Liberase PI and low-temperature isolation technique. However, overdigestion of islets during the isolation remains to be overcome. Advance in porcine islet isolation technique will in the future make the porcine islet xenotransplantation a reality for the cure of diabetes mellitus.

Section snippets

Donor animals

All pigs were purchased from an official supplier and housed until their body weight was over 200 pounds in accord with the Guideline for Laboratory Animal. Pigs were sacrificed at a slaughterhouse by induction of electric shock. Blood was collected via a cervical vein immediately after sacrifice.

Pancreas preparation

Splenic, duodenal, and connecting lobes of the pancreas were procured ex vivo within 10 minutes of warm ischemia and placed into chilled preservation solution. The pancreas was divided into three

Results

Twenty pancreata were procured for islet isolation. Islet isolation was performed with modified Ricordi's method using a new enzyme Liberase PI. Quantitation of islet viability, insulin stimulation assay, intracellular insulin content/DNA assay, and in vivo transplantation into diabetic nude mice were done for quality control of the islets. These results were compared between a high-yield group (>2500 IEQ/g pancreas) and a low-yield group (<2500 IEQ/g pancreas). A sufficient amount of purified

Discussion

Pig islets seem to be a potential source of pancreas islet cells for transplantation.¹ First, pig insulin has been used in humans for patients with diabetes mellitus for a long time. Second, ethical problems may be smaller than any other species, because pigs are bred worldwide for meat. Third, pig donors can be selected to favor high yields by age, sex, timing, race,² and feeding materials.³ Last, the risk of transmission of xenotic pathogens to human recipients may be reduced by breeding

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Citation Excerpt :
Eurocollins solutions or HTK solutions were used for pancreas perfusion. Isolation and purification were performed as previously described.1 Briefly, the pancreas was divided into three parts, including the splenic, duodenal, and connecting tissue.
Adult porcine islet xenotransplantation into humans is greatly diminished by the difficulty to isolate islets because of their fragility. The goal of this study was to improve the efficacy of islet yields using endogenous trypsin inhibitor and histidine-tryptophan-ketoglutarate (HTK) perfusate.
We compared two porcine islet isolation protocols: Eurocollins solution for in situ pancreas perfusion without use of an endogenous trypsin inhibitor versus HTK solution including endogenous trypsin inhibitor for pancreas perfusion.
Endogenous trypsin inhibitor and HTK strategies significantly improved total islet yield, recovery, and islet index after purification (P < .05), whereas unpurified islet yield did not increase. An average of 228,000 ± 95,000 islet equivalents (IEQ) (n = 20) purified islets were obtained in the first group compared with 115,000 ± 56,000 IEQ (n = 18) in the second group. The average islet index was significantly increased in the first group compared with the second group before and after purification: before: 0.28 versus 0.49 versus after: 0.25 versus 0.4 (P < .05). At this time, islet purity, viability, and stimulation index did not show a significant difference between groups.
Our study showed that endogenous trypsin inhibitor and HTK strategies significantly improved purified islet isolation efficacy because of reduction of islet fragility.
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: Supported by grants from Asan Institute for Life Science (2004-211).

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