An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells.
Here we describe a simple and efficient human embryonic stem (ES) and induced pluripotent stem (iPS) cells cryopreservation protocol. This protocol involves the use of Rho-associated kinase (ROCK) inhibitor, Y-27632, for the feeder-free dissociated cells. The addition of ROCK inhibitor to both pre- and post-thaw culture media enhanced the cloning efficiency. The presence of Y-27632 in Matrigel further increased the cloning efficiency. As compared with other available protocols for human ES and iPS cells cryopreservation, our protocol differs in the technical simplicity, high cloning efficiency and post-thawing passaging. We believe that this protocol could be a generally applicable and robust platform for feeder-free cryopreservation and the expansion of present and future applications of human ES and iPS cells. The treatment with ROCK inhibitor, cell harvesting and the freezing-thawing process usually takes about 2 h excluding overnight incubation at -80 degrees C.
Baharvand
Hossein
H
Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. Baharvand@RoyanInstitute.org
Salekdeh
Ghasem Hosseini
GH
Taei
Adeleh
A
Mollamohammadi
Sepideh
S
D000577:Amides; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D003094:Collagen; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D007797:Laminin; D047428:Protein Kinase Inhibitors; D011509:Proteoglycans; D011725:Pyridines; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
D000577:Amides; D003470:Culture Media; D004338:Drug Combinations; D007797:Laminin; D047428:Protein Kinase Inhibitors; D011509:Proteoglycans; D011725:Pyridines; C059485:matrigel; C108830:Y 27632; D003094:Collagen; D054460:rho-Associated Kinases