Increased islet viability by addition of beraprost sodium to collagenase solution.
The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 nM was added to the collagenase solution. After purification, the islet yield was 434,561 +/- 35.691 islet number expressed as 150 microm equivalent size (IEQ)/pancreas or 8,799 +/- 345 IEQ/g of pancreas in the BPS group and 349,987 +/- 52,887 IEQ/pancreas or 7,998 +/-1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 +/- 0.7% in the BPS group and 82.0 +/-0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number x % viability) was 384,586 +/- 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 +/- 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 +/- 38.5 versus 92.3 +/- 25.6 microU/IEQ; P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves beta cell function.
Arita
S
S
Department of Surgery, UCLA School of Medicine and Veterans Affairs Medical Center/West Los Angeles, 90073, USA.
Une
S
S
Ohtsuka
S
S
Kawahara
T
T
Kasraie
A
A
Smith
C V
CV
Mullen
Y
Y
D000818:Animals; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured / Q000737:chemistry / Q000378:metabolism / Q000637:transplantation; D002499:Centrifugation, Density Gradient; D017364:Collagenases / Q000494:pharmacology*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004285:Dogs; D011464:Epoprostenol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000379:methods*; D012996:Solutions; D013696:Temperature
D003451:Cryoprotective Agents; D007328:Insulin; D012996:Solutions; C048081:beraprost; D011464:Epoprostenol; D017364:Collagenases