Effects of synthetic antifreeze glycoprotein analogue on islet cell survival and function during cryopreservation.
The antifreeze glycoprotein (AFGP), found in the blood of polar fish, is known to prevent ice crystal growth and to depress the freezing temperature, which may in turn protect tissues from freezing injury. The chemical synthesis of AFGP is an attractive alternative to its difficult isolation from natural sources, and this would permit quality control and mass production. In spite of recent success in islet transplantation for the treatment of type 1 diabetes mellitus, existing methods for the long-term preservation of islets are considered to be suboptimal and inadequate, which indicates the need for the development of improved methods. Rat islets were isolated from male Wistar rats, using intraductal collagenase distention, mechanical dissociation, and Ficoll-Conray gradient purification. Islets were cultured overnight and then cryopreserved in RPMI1640 in the presence of dimethyl sulfoxide (Me2SO) and 10% FCS with various concentrations of syAFGP, followed by slow cooling (0.3 degrees C/min) and rapid thawing (200 degrees C/min) as described by Rajotte. The freezing process was observed by cryomicroscopy. Islet recovery post-cryopreservation was 85.0 +/- 6.2% with syAFGP and 63.3 +/- 14.2% without syAFGP, both compared with the pre-cryopreservation counts (P < 0.05). The in vitro islet function measured by insulin release was equivalent to a static stimulation index of 3.86+/-0.43 for the islets that were frozen-and-thawed with syAFGP, compared to 2.98 +/- 0.22 without syAFGP (P < 0.05). At a concentration of around 500 microg/ml syAFGP, a strong attenuation of ice crystal growth and formation was observed by cryomicroscopy and these ice crystals did not cause cryoinjury. In conclusion, the attenuation of ice crystallization by syAFGP improves islet survival and function following cryopreservation and thawing.
Matsumoto
Shuichiro
S
First Department of Surgery, Hokkaido University School of Medicine, N15-W7, Kita-ku, Sapporo 060-8638, Japan. matsu@med.hokudai.ac.jp
Matsusita
Michiaki
M
Morita
Tsunehiko
T
Kamachi
Hirofumi
H
Tsukiyama
Shusaku
S
Furukawa
Yoshinori
Y
Koshida
Shuhei
S
Tachibana
Yuki
Y
Nishimura
Shin-Ichiro
S
Todo
Satoru
S
D000818:Animals; D021301:Antifreeze Proteins / Q000378:metabolism / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects*; D020285:Cryoelectron Microscopy; D015925:Cryopreservation / Q000379:methods*; D004789:Enzyme Activation; D004912:Erythrocytes / Q000378:metabolism; D005615:Freezing; D005690:Galactose / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar
D021301:Antifreeze Proteins; D005690:Galactose