title: Efficient method for slow cryopreservation of human embryonic stem cells in xeno-free conditions.
abstract: 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.
authors.affiliation: Valencia Stem Cell Bank, Centro de Investigación Principe Felipe, Valencia University, Valencia, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Valbuena
authors.forename: Silvia
authors.initials: S
authors.lastname: Sánchez-Luengo
authors.forename: Amparo
authors.initials: A
authors.lastname: Galán
authors.forename: Eva
authors.initials: E
authors.lastname: Sánchez
authors.forename: Eva
authors.initials: E
authors.lastname: Gómez
authors.forename: M Eugenia
authors.initials: ME
authors.lastname: Poo
authors.forename: Verónica
authors.initials: V
authors.lastname: Ruiz
authors.forename: Olga
authors.initials: O
authors.lastname: Genbacev
authors.forename: Ana
authors.initials: A
authors.lastname: Krtolica
authors.forename: Antonio
authors.initials: A
authors.lastname: Pellicer
authors.forename: Rubén
authors.initials: R
authors.lastname: Moreno
authors.forename: Carlos
authors.initials: C
authors.lastname: Simón
mesh_terms: 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
chemical_list: D003451:Cryoprotective Agents; D017931:DNA Primers; D004121:Dimethyl Sulfoxide