An effective serum- and xeno-free chemically defined freezing procedure for human embryonic and induced pluripotent stem cells.
BACKGROUND
Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) bear a great potential in regenerative medicine. In addition to optimized clinical grade culture conditions, efficient clinical grade cryopreservation methods for these cells are needed. Obtaining good survival after thawing has been problematic.


METHODS
We used a novel, chemically defined effective xeno-free cryopreservation system for cryostorage and banking of hESCs and iPSCs. The earlier established slow freezing protocols have, even after recent improvements, resulted in low viability and thawed cells had a high tendency to differentiate. The medium is a completely serum and animal substance free product containing dimethylsulfoxide, anhydrous dextrose and a polymer as cryoprotectants. The cells were directly frozen at -70 degrees C, without a programmed freezer.


RESULTS
The number of frozen colonies versus the number of surviving colonies differed significantly for both HS293 (chi(2) = 9.616 with one degree of freedom and two-tailed P = 0.0019) and HS306 (chi(2) = 8.801 with one degree of freedom and two-tailed P = 0.0030). After thawing, the cells had a high viability (90-96%) without any impact on proliferation and differentiation, compared with the standard freezing procedure where viability was much lower (49%). The frozen-thawed hESCs and iPSCs had normal karyotype and maintained properties of pluripotent cells with corresponding morphological characteristics, and expressed pluripotency markers after 10 passages in culture. They formed teratomas containing tissue components of the three germ layers.


CONCLUSIONS
The defined freezing-thawing system described here offers an excellent simple option for banking of hESCs and iPSCs.
Holm
Frida
F
Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K57, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden.
Ström
Susanne
S
Inzunza
José
J
Baker
Duncan
D
Strömberg
Anne-Marie
AM
Rozell
Björn
B
Feki
Anis
A
Bergström
Rosita
R
Hovatta
Outi
O
D001483:Base Sequence; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D016895:Culture Media, Serum-Free; D017931:DNA Primers / Q000235:genetics; D004121:Dimethyl Sulfoxide; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007621:Karyotyping; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011108:Polymers; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013724:Teratoma / Q000209:etiology
D003451:Cryoprotective Agents; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011108:Polymers; D012333:RNA, Messenger; D005947:Glucose; D004121:Dimethyl Sulfoxide