Immersion of pancreas for 24 hours in collagenase solution at 4°C in Langerhans islets isolation [Abstract in English]

Luciano Passamani Diogo, David Saitovitch, Laura Fuchs Bahlis, Cínthia Fonseca O'Keeffe

Abstract


Aims: To test whether the immersion of the pancreas in solutions of 1 or 2 mg/mL of collagenase in 4°C for 24 hours, for the isolation of Langerhans islets, rises the yield of islets/grams of pancreatic tissue.
Methods: Experimental study with mouses, performed in the Laboratory of Nephrology of the Instituto de Pesquisas Biológicas do Hospital São Lucas da PUCRS, Porto Alegre, RS. After the animals have been sacrified under anesthesia, the pancreas were removed and divided in four groups, according the technique used for isolating the islets. A) collagenase 1mg/mL, in 4ºC for 24 hours and heating for 39ºC for 15 minutes. B) collagenase 2mg/mL with the same previous described steps. C) collagenase 1mg/mL and heating of the solution in the same day, in 39ºC for 15 minutes. D) collagenase 2mg/mL and heating of the solution in the same day, in 39ºC for 15 minutes. We verified the viability of the islet through the trypan blue test.
Results: The median numbers of isolated islets in the groups A, B, C and D were 9142, 8285, 2813 e 3199, respectively. Kruskal-Wallis test showed significant difference, with the value of H = 17,44 with a = 0,01, and in the comparison between the groups, there was no difference in the solutions of collagenase with concentrations of 1 and 2 mg/dL. Groups with immersion of pancreatic tissue in collagenase solution for 24 hours had three times more islets when compared to groups submitted to immediate digestion, according to Dunn test with a <0,05. The trypan blue test showed viability higher than 95% in all groups.
Conclusions: We suggest that the immersion in solution of collagenase for longer time improves the process of digestion of pancreatic tissue, rising the yield of islets/ grams of pancreatic tissue. Different concentration between
the collagenase solutions did not affect the final result.
KEY WORDS: ISLETS OF LANGERHANS TRANSPLANTATION; CULTURE TECHNIQUES; COLLAGENASES; ORGAN PRESERVATION SOLUTIONS; EPIDEMIOLOGY, EXPERIMENTAL; PANCREAS; MICE; TISSUE PRESERVATION; COOLING.



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