Significant progress in porcine islet mass isolation utilizing liberase HI for enzymatic low-temperature pancreas digestion.
BACKGROUND
Frequent success in human islet isolation is prevented by the large variability of scarce organ donors; this favors the future utilization of pigs as donors for clinical islet xenotransplantation. Porcine-specific difficulties of islet isolation are attributed to the intrinsic fragility of islets during pancreas digestion.


METHODS
To preserve islet integrity during efficient pancreas dissociation, porcine pancreata (n=48) were distended after cold storage with cold University of Wisconsin solution containing Liberase HI and digested at 24-28 degrees C using digestion-filtration. Pancreata distended with University of Wisconsin solution containing well-proven crude collagenase and digested at 32-34 degrees C served as controls (n=46). Monolayer Ficolldiatrizoate gradient purification was performed in a Cobe 2991.


RESULTS
Purified yield of islet equivalents per pancreas (mean+/-SEM) was almost doubled by Liberase HI compared with crude collagenase (526,480+/-46,560 vs. 270,270+/-19,420; P < 0.0001) and also significantly increased comparing islet equivalents per gram of pancreas (4,210+/-320 vs. 2,640+/-245; P=0.0004). Islet integrity was better preserved during Liberase HI digestion compared with crude collagenase digestion as indicated by isolation index (2.1+/-0.1 vs. 1.4+/-0.1; P<0.0001). Purity, viability, and in vitro function of islets did not differ between experimental groups. Preserved in vivo function of islets isolated by Liberase HI was demonstrated after subcapsular transplantation into 16 diabetic nude rats.


CONCLUSIONS
If the problems related to xenograft rejection and xenosis could be solved, low-temperature digestion of porcine pancreata using Liberase HI could serve as an essential prerequisite for successful 1:1 xenotransplantation of pig islets into type 1 diabetic human recipients.
Brandhorst
H
H
Third Medical Department, Justus-Liebig-University, Giessen, Germany.
Brandhorst
D
D
Hering
B J
BJ
Bretzel
R G
RG
D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D002469:Cell Separation; D002470:Cell Survival / Q000502:physiology; D017364:Collagenases / Q000378:metabolism; D005978:Glutathione; D006801:Humans; D007328:Insulin; D007515:Islets of Langerhans / Q000166:cytology*; D019188:Organ Preservation Solutions; D011887:Raffinose; D013552:Swine; D013820:Thermolysin / Q000378:metabolism
D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose