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.

journal_name

Reprod Biomed Online

authors

Valbuena D,Sánchez-Luengo S,Galán A,Sánchez E,Gómez E,Poo ME,Ruiz V,Genbacev O,Krtolica A,Pellicer A,Moreno R,Simón C

doi

10.1016/s1472-6483(10)60302-1

subject

Has Abstract

pub_date

2008-07-01 00:00:00

pages

127-35

issue

1

eissn

1472-6483

issn

1472-6491

pii

S1472-6483(10)60302-1

journal_volume

17

pub_type

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