LAP-1 cold preservation solution for isolation of high-quality human pancreatic islets.
The most critical factors that affect the outcome of clinical pancreatic islet transplantation are the number and quality of donor islets available for transplantation. Toward this goal, we attempted to obtain islets that are both of better quality and higher number than are obtainable by the islet-isolation process that is now widely used. We paid special attention to two critical components of the isolation procedure: minimizing the exposure of pancreatic tissue and freed islets to warm enzyme solution, and development of a preservation solution suitable for islets during cold storage of digested pancreatic tissue-free islets. For this purpose, we developed both a two-step procedure for pancreas digestion and a new cold preservation solution, the LAP-1 solution (Los Angeles preservation solution 1). In this study, we evaluated the effect of four preservation solutions by storing digested pancreatic tissues on ice for 90 min. After the cold storage, islets were purified on three layers of Euro-Ficoll solutions in a 50-ml tube, and the islet yield, viability, and function were determined. These experiments were performed by using samples from 10 consecutive islet isolations. Results with LAP-1, original University of Wisconsin solution (oUW), and modified UW solution (mUW;UW without hydroxyethyl starch) were compared with those obtained with Hank's balanced salt solution (HBSS). The islet yield was significantly higher in the LAP-1 and mUW groups as compared with the HBSS group (p < 0.01). The islet purity was significantly better in the LAP-1, oUW, and mUW groups than the HBSS (p < 0.001). The islet viability was lowest in the HBSS group immediately after purification (vs. LAP-1, oUW, and mUW, p < 0.05) and further decreased during culture (p < 0.01). Both the number and viability of cultured islets were the highest with LAP-1 solution but without statistical significance between mUW and oUW. Electron microscopic examination showed only slight damage to cell membranes immediately after purification of islets stored in LAP-1 solution and their complete recovery within 1-2 days of culture. These islets also exhibited normal insulin responses to high glucose by static incubation and perifusion assays.
Kenmochi
T
T
Department of Surgery, UCLA School of Medicine, USA.
Miyamoto
M
M
Sasaki
H
H
Une
S
S
Nakagawa
Y
Y
Moldovan
S
S
Benhamou
P Y
PY
Brunicardi
F C
FC
Tanaka
H
H
Mullen
Y
Y
D000293:Adolescent; D000328:Adult; D002452:Cell Count; D002469:Cell Separation; D002470:Cell Survival; D003080:Cold Temperature; D046508:Culture Techniques; D004187:Disaccharides; D005260:Female; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000502:physiology* / Q000648:ultrastructure; D016381:Islets of Langerhans Transplantation; D008297:Male; D008353:Mannitol; D008854:Microscopy, Electron; D008875:Middle Aged; D014021:Tissue Preservation
D004187:Disaccharides; D007328:Insulin; C091004:LAP-1 solution; D008353:Mannitol