Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.
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.
Goto
Masafumi
M
Department of Radiology, Oncology and Clinical Immunology, Division of clinical Immunology, University Hospital, Uppsala, Sweden. gotokichi@aol.com
Eich
Torsten M
TM
Felldin
Marie
M
Foss
Aksel
A
Källen
Ragnar
R
Salmela
Kaija
K
Tibell
Annika
A
Tufveson
Gunnar
G
Fujimori
Keisei
K
Engkvist
Margareta
M
Korsgren
Olle
O
D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation; D016015:Logistic Models; D008875:Middle Aged; D015999:Multivariate Analysis; D014019:Tissue Donors; D020858:Tissue and Organ Harvesting / Q000379:methods*; D009927:Tissue and Organ Procurement