Crucial role of pancreatic ductal collagenase injection for isolation of pancreatic islets.
We have been stressing the advantage of stationary digestion because of its simplicity and reproducible high yields of viable islets. In the present study optimal conditions of stationary in vitro collagenase digestion in mice and rats were examined. We also compared two possible routes for collagenase injection; ductal (PD) and portal venous (PV) based on subsequent islet yield and ability to reverse diabetes in rats. Three parameters which affect the quality of digestion are 1) collagenase concentration, 2) incubation time and 3) digestion temperature. Suitable conditions were easily determined and reproducible high yield of islets could be consistently obtained. The islets from one mouse pancreas (approximately 200 islets) could consistently restore normoglycemia in one STZ-induced diabetic mouse and the islets from one rat pancreas (500-600 islets) can restore normoglycemia in 5-6 STZ-induced diabetic mice within a couple of days. Islet yield in the PD method was greater than that in the PV method, and insulin release from PD islets in response to high glucose was well preserved after 24 hours of culture when compared to PV islets. The ability to restore normoglycemia in STZ-induced diabetic mice was well preserved when transplanting 100 PD islets as compared with the same number of PV islets. The PD islets revealed a well preserved structure with healthy endocrine cells, while the PV islets showed a dilated capillary network and distorted endocrine cell continuity. Histological examination of digested tissue following PD injection showed the complete destruction of pancreatic exocrine tissue, as well as mechanical separation and digestion of interstitial tissue between the islets and exocrine tissue, with the islet being preserved selectively intact.(ABSTRACT TRUNCATED AT 250 WORDS)
Gotoh
M
M
Second Department of Surgery, Osaka University Medical School, Japan.
Ohzato
H
H
Porter
J
J
Maki
T
T
Monaco
A P
AP
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D002452:Cell Count; D002469:Cell Separation; D002470:Cell Survival / Q000502:physiology; D066298:In Vitro Techniques; D007267:Injections; D007275:Injections, Intravenous; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D016381:Islets of Langerhans Transplantation / Q000379:methods* / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D003012:Microbial Collagenase / Q000008:administration & dosage; D010183:Pancreatic Ducts; D011169:Portal Vein; D051381:Rats; D011919:Rats, Inbred Strains
D001786:Blood Glucose; D007328:Insulin; D003012:Microbial Collagenase