Successful human islet isolation utilizing recombinant collagenase.
The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 +/- 0.8 vs. 85.6 +/- 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.
Brandhorst
Heide
H
Third Medical Department, Justus-Liebig-University of Giessen, Rodthohl 6, 35385 Giessen, Germany. heide.brandhorst@innere.med.uni-giessen.de
Brandhorst
Daniel
D
Hesse
Friederike
F
Ambrosius
Dorothee
D
Brendel
Mathias
M
Kawakami
Yoshiyuki
Y
Bretzel
Reinhard G
RG
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D017364:Collagenases; D006801:Humans; D007202:Indicators and Reagents; D007511:Ischemia; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D009929:Organ Size; D010179:Pancreas / Q000033:anatomy & histology; D010477:Perfusion / Q000379:methods; D011994:Recombinant Proteins; D013370:Subrenal Capsule Assay; D013820:Thermolysin; D020858:Tissue and Organ Harvesting / Q000379:methods*; D014183:Transplantation, Heterologous
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin