Vitrification of encapsulated hepatocytes with reduced cooling and warming rates.
We have used microencapsulated hepatocytes as model to develop a method of vitreous cryopreservation of large quantities of cell-containing constructs. The method included a pre-equilibration procedure in which the amount of penetrating cryoprotectant was gradually increased by 15% in each step. The optimal vitrification solution consists of 40% ethylene glycol and 0.6M sucrose. The concentration of 1M sucrose used for the first dilution solution with subsequent decrease of sucrose concentration to 0.7 M sucrose and by 0.2-0.15M for each subsequent step. This sucrose dilution procedure had no adverse effect on cell functions. Three cooling rates (400 degrees C/min and above) and three warming rates (650 degrees C/min and above), in combination with the proposed vitrification solution, were equally effective. The optimization of the procedure and solutions allow microencapsulated hepatocytes to be preserved with almost 100% retention of cell functions and no detectable damage to the fragile microcapsules. The de-linking of the cooling/warming rates with the effectiveness of vitrification potentially paves the way for large scale cryopreservation of complex tissue engineered constructs.
Kuleshova
L L
LL
Department of Physiology, National University Medical Institutes, National University of Singapore, Block MD11, 10 Medical Drive, Singapore 117597. phskl@nus.edu.sg
Wang
X W
XW
Wu
Y N
YN
Zhou
Y
Y
Yu
H
H
D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D019855:Ethylene Glycol; D022781:Hepatocytes; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013395:Sucrose; D013696:Temperature; D014508:Urea / Q000378:metabolism
D013395:Sucrose; D014508:Urea; D003577:Cytochrome P-450 Enzyme System; D019855:Ethylene Glycol