While these standard methods are efficient for the cryopreservation of mouse ES cells ( Robertson, 1987), it has been observed that the survival of undifferentiated human ES cells following slow-rate freezing is very poor, and most of the cells either differentiate or die.Р”Р„s embryonic stem cells originate from the pluripotent cells of the blastocyst and retain in culture the properties of these cells such as morphology, gene expression and pluripotentiality, it has been postulated that methods developed specifically for the cryopreservation of blastocysts might be also efficient with human ES cells. Slow-rate freezing and rapid thawing methods are most commonly used for the cryopreservation of cell lines ( Freshney, 1994). Lastly, effective freezing and thawing technologies will allow effortless transfer of the cells between research centres, promoting scientific collaboration and facilitating widespread use of the cells for research and clinical applications.

Moreover, efficient cryopreservation will be essential if human ES cell banks are to be established ( Gearhart, 1998).

Effective freezing and thawing techniques would enable the efficient preservation of stocks of early passage cells, as well as the conservation of specific clones that are developed from the original cell lines such as genetically modified clones. In this context, an efficient cryopreservation method would be highly valuable for the development and widespread use of these cell lines. In order to exploit this remarkable potential of human ES cells, improvement of currently used technologies for handling and manipulating the cells is required. Given the self-renewal potential of ES cells, combined with their capability to differentiate into any cell type, these cells can potentially provide an unlimited source of cells for transplantation therapy ( Keller and Snodgrass, 1999). They may be used for the development of new drugs and for the discovery of new growth and differentiating factors that may have therapeutic applications. Human ES cells provide a powerful in-vitro model for the study of early human embryology. ES cell lines have been derived recently from human blastocysts ( Thomson et al., 1998 Reubinoff et al., 2000) and are expected to have far-reaching applications in the areas of regenerative medicine, pharmacology and basic scientific research. These cell lines can be propagated indefinitely in vitro, yet still retain a normal karyotype and the capacity for differentiation into a wide variety of somatic tissues representing progeny of the three embryonic germ layers. CONCLUSIONS: Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.Р”В¬ryopreservation, human embryonic stem cells, vitrification IntroductionР”В®mbryonic stem (ES) cell lines are derived from the pluripotent cells of the inner cell mass of the blastocyst. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers.

The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. METHODS AND RESULTS: All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. BACKGROUND: Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo.

Colony survival straw