Ultra-Fast Vitrification of Murine Embryonic Stem Cells Using a Low Concentration of Cryoprotectants

Effective cryopreservation of important mammalian cells and their derivatives is critical to the success of cell based medicine in 21^st century [1]. There are currently two approaches to achieve cryopreservation of mammalian cells: conventional slow freezing and vitrification without ice formation. Although conventional slow freezing only requires a low relatively nontoxic concentration of cryoprotectants (1–2 M), it is always associated with cell injury due to ice formation and freeze concentration (i.e., solute effect) [2]. Cryopreservation by vitrification avoids ice formation all together. Existing protocols for vitrification, however, require a very high concentration of cryoprotectants (CPAs, generally more than 4M) that is usually toxic to most mammalian cells [3]. Therefore, it is of great interest to achieve vitrification using a low nontoxic concentration of cryoprotectants, which combines the advantages of the existing slow freezing and vitrification approaches while avoiding their shortcomings. In this study, we report the successful vitrification of murine embryonic stem (ES) cells at a low nontoxic level of cryoprotectants utilizing a thin walled (10μm) quartz microcapillary (outer diameter, 200μm). The ES cells post-vitrification retained high immediate viability, attachment efficiency and similar proliferation characteristic to fresh ES cells. Expression of markers characteristic to the ES cells suggests the ES cells retained the undifferentiated properties of pluripotent cells post-vitrification.