Geraldine Parnaud1, Vanessa Lavallard1, Raphael Meier1, Bedoit Bedat1, Domenico Bosco1, Thierry Berney1
1Cell Isolation and Transplantation Center, Division of Surgical Research, Geneva University Hospital and University of Geneva, Geneva, Switzerland
Geraldine Parnaud, Switzerland
BACKGROUND: The quality of the enzymes used to dissociate the pancreas is critical for successful islet isolation. Despite the safety of the enzymes is certified, enzyme production still requires contact with some animal products. To abolish the risk of contamination of animal derived adventitious agents, animal-free collagenase (AF-1) and neutral protease (NP AF) were developed by SERVA. In this study, we assessed the impact of AF-1 and NP AF on islet isolation outcome.
METHODS: We report the results of 10 islet isolations from pancreases digested with SERVA Collagenase NB 1 Premium Grade with Neutral Protease NB High Active Grade (n=5) or with SERVA Collagenase AF-1 with Neutral Protease AF (n=5). We used an enzyme mixture composed of >2000 U of collagenase and 160 U of neutral protease. Islet isolation outcomes and islet quality were determined from the two enzyme preparations and statistically compared using Student’s test analysis.
RESULTS: No significant differences in donor-related factors were found between the groups. There were also no significant difference in time (NB 1 vs. AF-1 : 17.8 ± 4.0 vs. 17.2 ± 1.1 min) and efficacy of digestion (NB1 vs. AF-1: 89 ± 8% vs. 91 ± 2%). Using Collagenase AF-1 islet products encountered release criteria for transplantation from all of the 5 pancreases digested and from 3 of 5 pancreases when using Collagenase NB 1. Islet yields were similar between the two groups with 354110±164470 and 364510±134490 total IEQ/transplantation and 4550±2562 and 4153±3020 IEQ/g pancreas for NB 1 and AF-1 respectively. Furthermore beta-cell secretory function and viability were similar for the two groups.
CONCLUSION: The enzyme mixture composed of animal free collagenase (AF-1) with animal free neutral protease recovers similar islet yield than the Collagenase NB 1 Premium Grade while retaining islet quality and function. Thus, these enzymes represent a promising and safe new alternative to the enzymes which are currently used for islet isolation.
|2013-09-27||12:45 - 13:30||Non-CME Session||Automated human islet culture with continuous renewal of medium using PRISM technology||Carmel Room|
|2013-09-26||15:30 - 16:30||Rapid-Fire 7 on Technical Aspects of Islet Isolation and Culture||Human islet isolation using animal-free collagenase and neutral protease||Big Sur Room|
|2013-09-25||11:00 - 12:30||Parallel Session 4 on Islet Procurement, Preservation and Cell Biology||Cadherin engagement improves insulin secretion of single human beta-cells||Big Sur Room|