Efficient long-term cryopreservation of pluripotent stem cells at -80 °C.
Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2), because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures, e.g. -80 °C. This instability leads to ice recrystallization, causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (~-80 °C) of laboratory deep freezers. Moreover, a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at -80 °C for periods that extend up to at least one year, with the post-thaw viability, plating efficiency, and full retention of pluripotent phenotype comparable to that achieved with LN2storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2.
Division of Animal Sciences and Bond Life Sciences Center; University of Missouri, Columbia, MO 65211, USA.
Ye
Y
Yuan
Division of Animal Sciences and Bond Life Sciences Center; University of Missouri, Columbia, MO 65211, USA.
Ying
Y
Yang
Division of Animal Sciences and Bond Life Sciences Center; University of Missouri, Columbia, MO 65211, USA.
Yuchen
Y
Tian
Division of Animal Sciences and Bond Life Sciences Center; University of Missouri, Columbia, MO 65211, USA.
Jinkyu
J
Park
Division of Animal Sciences and Bond Life Sciences Center; University of Missouri, Columbia, MO 65211, USA.
Aihua
A
Dai
Division of Animal Sciences and Bond Life Sciences Center; University of Missouri, Columbia, MO 65211, USA.
R Michael
RM
Roberts
Department of Mechanical &Aerospace Engineering; University of Missouri, Columbia, MO 65211, USA.
Yang
Y
Liu
Department of Mechanical &Aerospace Engineering; University of Missouri, Columbia, MO 65211, USA.|Comparative Medicine Center, University of Missouri, Columbia, MO 65211, USA.
Xu
X
Han
D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000737:chemistry / Q000494:pharmacology; D005362:Ficoll / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D013552:Swine
D003451:Cryoprotective Agents; D005362:Ficoll; D004121:Dimethyl Sulfoxide
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