Maintenance of glucose-sensitive insulin secretion of cryopreserved human islets with University of Wisconsin solution and ascorbic acid-2 glucoside.
Normal human islet cells are an ideal source for pancreas-targeted cell therapies, but the availability of human donor pancreata for islet isolation is severely limited. To effectively utilize such scarce donor organs for cell therapies, it is crucial to develop an excellent isolation, effective cryopreservation, and efficient gene transfer techniques for the transportation of isolated cells. In the present study, we investigate the effect of University of Wisconsin (UW) solution and ascorbic acid-2 glucoside (AA2G) on the cryopreservation of human islets. We also evaluate the gene transfer efficiency of a lentiviral vector expressing the E. coli LacZ gene, Lt-NLS/LacZ, in human islets. Human islets were isolated with a standard digestion method at the University of Alberta. Isolated islets were transported to Japan for 40 h and then subjected to cryopreservation experiments. The following preservation solutions were tested: UW solution with 100 micro g/mL of AA2G, UW solution, 100% fetal bovine serum (FBS), and CMRL supplemented with 10% FBS. Following three months of cryopreservation, the islets were thawed and analyzed for viability, glucose-sensitive insulin secretion, proinsulin gene expression profile, and in vivo engraftment. The islets were also subjected to monolayer formation with 804G-cell-line-derived extracellular matrix (ECM), followed by Lt-NLS/LacZ transduction. The viability, morphology, glucose-sensitive insulin secretion, proinsulin gene expression, and monolayer formation efficiency of the thawed cryopreserved islets are significantly better maintained by the use of UW solution. When AA2G (100 microg/mL) is combined with UW, such parameters are further improved. The adequate engraftment of UW + AA2G-cryopreserved human islets is achieved in the liver of nude mice. Efficient Lt-NLS/LacZ transduction is identified in monolayered islets cryopreserved with UW solution with AA2G. The present work demonstrates that the combination of UW solution with AA2G (100 microg/mL) would be a useful cryopreservation means for human islets. Human islets monolayer-cultured with 804G-derived ECM are efficiently transduced with a lentiviral vector Lt-NLS/LacZ.
Arata
Takashi
T
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Okitsu
Teru
T
Fukazawa
Takuya
T
Ikeda
Hideaki
H
Kobayashi
Kazuya
K
Yong
Chen
C
Kosaka
Yoshikazu
Y
Narushima
Michiki
M
Matsuoka
Junji
J
Yamamoto
Itaru
I
Tanaka
Noriaki
N
Lakey
Jonathan R T
JR
Kobayashi
Naoya
N
D000241:Adenosine / Q000494:pharmacology*; D000493:Allopurinol / Q000494:pharmacology*; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002470:Cell Survival; D017690:Cell Transplantation; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D004247:DNA / Q000032:analysis; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D005978:Glutathione / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D008819:Mice, Nude; D019188:Organ Preservation Solutions; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D011887:Raffinose / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D014021:Tissue Preservation / Q000379:methods; D014183:Transplantation, Heterologous
D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D012313:RNA; D000493:Allopurinol; D004247:DNA; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose; D001205:Ascorbic Acid