Application of the two-layer method on pancreas digestion results in improved islet yield and maintained viability of isolated islets.
BACKGROUND
Oxygenation of the pancreas during preservation by the two-layer method (TLM) has shown beneficial effects in islet transplantation. Here, we apply this concept (oxygenation) to the isolation process.


METHODS
Rat pancreases were digested using four different methods. Pancreases were digested with preoxygenated perfluorocarbon (PFC) in group 2 and without it in group 1. Additionally, adenosine was included in the collagenase solution in subgroups B but not in subgroups A. Islet yields and viability were compared between groups.


RESULTS
Tissue oxygen tension in group 1 was essentially zero during digestion, but rapidly reached around 300 mm Hg and was maintained in group 2. The tissue adenosine triphosphate (ATP) level in rat pancreas just after laparotomy (control) was 4.2+/-0.7 micromol/g dry weight; after digestion, it was 0.12+/-0.03 micromol/g, 0.70+/-0.10 micromol/g, 0.30+/-0.18 micromol/g, and 2.90+/-0.80 micromol/g in groups 1A, 1B, 2A, and 2B, respectively. No significant differences were observed between group 2B and control (P=0.19). Islet yields (IEQ/pancreas) were 1600+/-400, 1400+/-400, 1300+/-400, and 2400+/-100 in groups 1A, 1B, 2A, and 2B, respectively. The islet yield of group 2B was significantly higher than other groups (P<0.05). The cure rate after transplanting 200 islets into athymic nude mice did not differ (80% in all groups). The stimulation indices in the four groups were also the same.


CONCLUSIONS
Tissue ATP levels after digestion were well maintained using TLM with adenosine digestion method. Consequently, greater numbers of islets could be retrieved. The new method was at least equivalent to islet function isolated by conventional method. Clinical study is therefore warranted.
Goto
Tadahiro
T
Division of Gastroenterological Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
Tanioka
Yasuki
Y
Sakai
Tetsuya
T
Terai
Sachio
S
Kamoda
Yasuhisa
Y
Li
Shiri
S
Tanaka
Tomohiro
T
Tsujimura
Toshiaki
T
Matsumoto
Ippei
I
Fujino
Yasuhiro
Y
Suzuki
Yasuyuki
Y
Kuroda
Yoshikazu
Y
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001786:Blood Glucose; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D005951:Glucose Tolerance Test; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000379:methods*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew
D001786:Blood Glucose; D000255:Adenosine Triphosphate; D000241:Adenosine; D010100:Oxygen