Cold preservation of islets in UW solution--with special reference to apoptosis.
BACKGROUND
Apoptosis progresses in cultured islets. Little is known with regard to apoptosis under cold preservation. We examined viability and function of islets in University of Wisconsin (UW) solution.


METHODS
Isolated rat islets were cultured overnight (overnight group) and further treated with 7-day culture in RPMI 1640 medium at 37 degrees C (culture group) or 7-day preservation in UW solution at 4 degrees C (preservation group). They were evaluated by glucose-stimulated insulin secretion test. Apoptosis was examined by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Expression of caspase mRNA and the ratio of Bax to Bcl-2 were evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR).


RESULTS
Islet recovery after 7 days was significantly lower in culture group than in preservation group (44.0 +/- 3.7% versus 75.0 +/- 4.9%, P < 0.05). The stimulation index in the culture group was significantly lower than in the overnight group (2.1 +/- 0.2 versus 4.1 +/- 0.4, P < 0.05). The apoptotic index in the culture group was significantly higher than both in the overnight group and in the preservation group (38.0 +/- 3.0% versus 10.8 +/- 2.0 and 27.0 +/- 4.0%, P < 0.05). Caspase 3, 8, and 9 mRNA in the culture group expressed more than in the other groups. Bax/Bcl-2 in the culture group was significantly lower than in the overnight group (3.2 +/- 0.66 versus 8.1 +/- 0.95, P < 0.05), suggesting that apoptosis had been already destined early after isolation.


CONCLUSIONS
The preservation group showed better recovery and function than the culture group. Apoptosis contributed to islet loss under culture and it was significantly suppressed under cold preservation.
Shirouzu
Yasumasa
Y
Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan. yasumasa@indigo.plala.or.jp
Gu
Yuanjun
Y
Koga
Mari
M
Sakurai
Tomonori
T
Qi
Meirigeng
M
Hiura
Akihito
A
Sumi
Shoichiro
S
Inoue
Kazutomo
K
D000241:Adenosine / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000235:genetics; D015925:Cryopreservation / Q000379:methods*; D005947:Glucose / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D008297:Male; D009924:Organ Culture Techniques; D019188:Organ Preservation Solutions / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D011887:Raffinose / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007328:Insulin; D019188:Organ Preservation Solutions; D012333:RNA, Messenger; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D020169:Caspases; D005978:Glutathione; D005947:Glucose; D000241:Adenosine; D011887:Raffinose