Cryoprotectants for the vitrification of corneal endothelial cells.
The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7kDa), dextran (MW 7kDa), chondroitin sulfate (CS, MW 18-30kDa), bovine serum albumin (MW 68kDa) and polyethylene glycol (MW 6kDa, 10kDa and 20kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4+/-2.1% (mean+/-SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.
Fan
Wen-Xia
WX
Dalian R&D Center for Stem Cell and Tissue Engineering, School of Chemical Engineering, Dalian University of Technology, Room 3-407, Life Science Building, Dalian 116024, China.
Ma
Xue-Hu
XH
Dalian R&D Center for Stem Cell and Tissue Engineering, School of Chemical Engineering, Dalian University of Technology, Room 3-407, Life Science Building, Dalian 116024, China. Electronic address: xuehuma@dlut.edu.cn.
Ge
Dan
D
Dalian R&D Center for Stem Cell and Tissue Engineering, School of Chemical Engineering, Dalian University of Technology, Room 3-407, Life Science Building, Dalian 116024, China.
Liu
Tian-Qing
TQ
Dalian R&D Center for Stem Cell and Tissue Engineering, School of Chemical Engineering, Dalian University of Technology, Room 3-407, Life Science Building, Dalian 116024, China.
Cui
Zhan-Feng
ZF
Department of Engineering Science, Oxford University, Parks Road, Oxford OX1 3PJ, UK.
D000818:Animals; D002241:Carbohydrates; D002417:Cattle; D002463:Cell Membrane Permeability; D002470:Cell Survival; D002478:Cells, Cultured; D002809:Chondroitin Sulfates; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004728:Endothelium, Corneal / Q000166:cytology*; D019855:Ethylene Glycol; D005947:Glucose; D008970:Molecular Weight
D002241:Carbohydrates; D003451:Cryoprotective Agents; D002809:Chondroitin Sulfates; D019855:Ethylene Glycol; D005947:Glucose