Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.
Transplantation
; 78(9): 1367-75, 2004 Nov 15.
Article
em En
| MEDLINE
| ID: mdl-15548977
ABSTRACT
BACKGROUND:
The procedure of human islet isolation needs further optimization and standardization. Here, we describe techniques to enhance enzymatic digestion and minimize mechanical forces during the digestion process. The isolation protocol has also been modified to meet current GMP (cGMP) standards. Moreover, the impact of donor- and process-related factors was correlated to the use of islets for clinical transplantation.METHODS:
One hundred twelve standardized consecutive islet isolations were evaluated. Metyltioninklorid and indermil (topical tissue adhesive) were applied to detect leakage of collagenase injected and to repair the damaged pancreatic glands. The effects of dye and glue were evaluated in terms of islet yield, islet function using the perifusion assay, and success rate of the isolation. To analyze key factors for successful isolations, both univariate and multivariate regression analysis were performed.RESULTS:
Both Metyltioninklorid and Indermil were effective to prevent leakage of enzyme solutions from the pancreatic glands. Both islet yield and success rate were higher when these tools were applied (4,516.1+/-543.0 vs. 3,447.7+/-323.5, P=0.02; 50.0% vs. 21.3%, P=0.02, respectively). No adverse effects on islet function or collagenase activity were observed. Multivariate regression analysis identified the maximal recorded amylase >100 U/L (P=0.026), BMI (P=0.03), and the use of catecholamine (P=0.04) as crucial donor-related factors. In addition, cold ischemia time (P=0.005), the dissection procedure using whole glands with duodenum (P=0.02), and the local procurement team (P=0.03) were identified as crucial isolation-related variables.CONCLUSIONS:
A standardized technique of islet isolation is presented applying novel means to improve enzymatic digestion and to meet cGMP standards.
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Base de dados:
MEDLINE
Assunto principal:
Ilhotas Pancreáticas
/
Coleta de Tecidos e Órgãos
Tipo de estudo:
Guideline
/
Prognostic_studies
Limite:
Humans
/
Middle aged
Idioma:
En
Ano de publicação:
2004
Tipo de documento:
Article