Efficient method for slow cryopreservation of human embryonic stem cells in xeno-free conditions.
An effective, consistent and xeno-free cryopreservation technique is crucial for any human embryonic stem cell (hESC) laboratory with future perspectives for clinical application. This study presents a new slow freezing-rapid thawing method in serum-free conditions that allows the cryopreservation of a large number of colonies without the use of a programmable freezer. To test its efficacy, this method has been compared with two established vitrification methods and applied to three different hESC lines (H9, VAL-3 and VAL-5). The method is based on an increasing concentration of dimethylsulphoxide (1.0, 1.2, 1.5 and 2.0 mol/l) with a slow or a rapid cooling system. Using this method, approximately 60 colonies per cryovial could be cryopreserved, the survival rate ranged between 15 and 68% depending on the cell line used, and the majority of the surviving colonies were grade A. Post-cryopreserved hESC have been cultured for 20 passages, re-cryopreserved and re-thawed with consistent results. After thawing, cells retained the inherent undifferentiated characteristics of hESC and growth rate curve, with a stable karyotype, telomerase activity and teratoma formation when injected into severe combined immunodeficient animals, which was comparable with the fresh lines. This method has been tested for 3 years in two different laboratories.
Valbuena
Diana
D
Valencia Stem Cell Bank, Centro de Investigación Principe Felipe, Valencia University, Valencia, Spain.
Sánchez-Luengo
Silvia
S
Galán
Amparo
A
Sánchez
Eva
E
Gómez
Eva
E
Poo
M Eugenia
ME
Ruiz
Verónica
V
Genbacev
Olga
O
Krtolica
Ana
A
Pellicer
Antonio
A
Moreno
Rubén
R
Simón
Carlos
C
D000818:Animals; D018920:Coculture Techniques; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D046149:Embryo Culture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D007621:Karyotyping; D051379:Mice; D016513:Mice, SCID; D013997:Time Factors
D003451:Cryoprotective Agents; D017931:DNA Primers; D004121:Dimethyl Sulfoxide