Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: A study using murine embryonic stem cells

Abstract

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz microcapillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1-2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

Preservation of Embryonic Stem Cells

“…As a result, the cell survival increases with the increase of cooling rate till it reaches 100%. Both CR SF and CR V are dependent on the cell type, the CPA type (propylene glycol has been reported to be superior to ethylene glycol in terms of the capability of vitrification (He et al 2008b)), and the CPA concentration. Of note, the damaging (both osmotic and metabolic) effect of an unusually high concentration of CPAs required by the conventional vitrification is not considered in the figure. www.intechopen.com ) and high (for vitrification, ) cooling rates.…”

“…Oftentimes, a mixture of multiple CPAs is used to reduce the cytotoxicity of the high CPA concentration required (Fuller 2004). In addition, large, membrane impermeable molecules such as sugars (typically sucrose and trehalose) have been used to dehydrate the cells somewhat before cooling and protect cell membrane from injury during cooling (Beattie et al 1997;He et al 2008b). Vitrification can be done without a specialized machine and the time required is much shorter than that for slow-freezing.…”

“…3 should be able to enhance vitrification of living cells encapsulated in the microcapsules at high cooling rates (e.g., > 10,000 o C/min). This is because it can not only depress ice formation and growth in the microcapsule but also prevent ice (if any) propagation into cells from the bulk solution where ice is usually formed first (because of its much bigger volume) (Berejnov et al 2006;Fahy et al 1987;Franks et al 1983;He et al 2008b;Karlsson et al 1994;Mazur et al 2005a;Mazur et al 2005b;Toner 1993;Toner et al 1990;Yavin and Arav 2007). This hypothesis is confirmed by a recent study where the C3H10T1/2 mouse mesenchymal stem cells encapsulated in ~100 µm alginate microcapsules were vitrified using a 400 µm, thin-walled quartz microcapillary at a low-CPA concentration (1.4 M DMSO) (Zhang et al 2010).…”

“…Vitrification can be done without a specialized machine and the time required is much shorter than that for slow-freezing. (He et al 2008b)) A comparison of the conventional French-type straw (top) used today for cell vitrification at an unusually high CPA concentration and the 200 µm (outer diameter), thin-walled (10 µm) quartz microcapillary (QMC, bottom) used to achieve ultrafast cooling to minimize the CPA concentration required for vitrification Another way to improve cell vitrification is to confine cells in a small space such as submilimeter (in diameter) sized liquid droplets of aqueous cell suspension (Berejnov et al 2006;Edd et al 2008;Franks et al 1983). A major disadvantage of using small liquid droplets to confine cells is that the droplets will merge with each other unless they are dispersed in an oil phase, which makes it difficult to retrieve cells from the droplets.…”

“…The unusually high (as high as 7 M) concentration of CPA required, however, can result in significant metabolic and osmotic injury to cells (Chen et al 2000;Chen et al 2001a;Fahy et al 2004a;Fowler and Toner 2005;Heng et al 2005;Hunt et al 2006). Consequently, it is necessary to use multiple steps of CPA loading/dilution and maintain a short exposure time (within a few minutes) to high concentration CPA in each step to minimize injury (Reubinoff et al 2001), which makes the procedure complicated, stressful, and particularly, difficult to control in that the time for the diffusion of CPAs into the cells to reach equilibrium usually takes at least 5-10 minutes (He et al 2008b;Heng et al 2005;Jain and Paulson 2006;Pedro et al 2005). In addition, a cocktail of various CPAs rather than one CPA has been commonly used to reduce the CPA toxicity.…”