Comparison of size, viability, and function of fetal pig islet-like cell clusters after digestion using collagenase or liberase.
Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 +/- 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 +/- 2 vs. 31 +/- 2 min). The size of ICCs was similar for both collagenase (83 +/- 0.5 microm) and Liberase (79 +/- 0.4 microm) as was the number of ICCs produced per pancreas (7,653 +/- 1,297 vs. 8,101 +/- 1,177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 +/- 1% vs. 76 +/- 1%, p < 0.05). Responsiveness to beta-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at I month after transplantation of 1,500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.
Georges
Pauline
P
Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, Australia.
Muirhead
Roslyn P
RP
Williams
Lindy
L
Holman
Sara
S
Tabiin
Muhammad Tani
MT
Dean
Sophia K
SK
Tuch
Bernard E
BE
D000818:Animals; D002469:Cell Separation / Q000379:methods; D048429:Cell Size; D002470:Cell Survival / Q000502:physiology*; D017364:Collagenases; D004731:Endotoxins / Q000032:analysis; D016332:Fetal Tissue Transplantation; D005333:Fetus; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000502:physiology; D051379:Mice; D013552:Swine; D013820:Thermolysin
D004731:Endotoxins; D007328:Insulin; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin