A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells.
BACKGROUND
An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation.


METHODS
We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632.


RESULTS
Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture.


CONCLUSIONS
This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.
Mollamohammadi
Sepideh
S
Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, PO Box 19395-4644, Tehran, Iran.
Taei
Adeleh
A
Pakzad
Mohammad
M
Totonchi
Mehdi
M
Seifinejad
Ali
A
Masoudi
Najmehsadat
N
Baharvand
Hossein
H
D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D003470:Culture Media; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005819:Genetic Markers; D006801:Humans; D007797:Laminin; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D011509:Proteoglycans
D003451:Cryoprotective Agents; D003470:Culture Media; D004338:Drug Combinations; D005819:Genetic Markers; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen