Preservation of Embryonic Stem Cells

Alginate Hydrogel Microencapsulation Inhibits Devitrification and Enables Large‐Volume Low‐CPA Cell Vitrification

“…Such an ice-seeding step is not used for vitrification and the thermodynamics of vitrification is typically non-equilibrium. As a result, for the cryomicroscopy study of IIF during low-CPA vitrification, ice crystals typically form in the extracellular solution at much lower temperatures (approximately −20 °C, Figures 2 and S2) than the thermodynamic equilibrium melting point (usually higher than −10 °C [2b] ) of the solutions and they are usually very fine as their growth is hampered by the slow diffusion of water molecules ( i.e. , diffusion-limited growth) at such deep subzero temperatures.…”

“…[1] Because continuous expansion of cells in vitro by culturing at 37 °C is expensive and time-consuming and may result in uncontrolled spontaneous differentiation of stem cells, banking living cells for future use by cryopreservation is an enabling technology to the eventual success of the burgeoning cell-based medicine. [2] Cell cryopreservation is achieved by first cooling cells to usually below −80 °C so that all the biophysical and biochemical activities in cells are arrested ( i.e. , the cells enter a state of suspended animation), followed by warming back for use at a desired future time.…”

“…For the former, cells are gradually dehydrated due to exosmosis driven by the elevated osmolality of extracellular solution when extracellular water transforms into ice during freezing at a slow cooling rate (typically less than 5 °C min −1 ), which is referred as freeze concentration and can cause significant damages to cells. [2b,5] On the other hand, conventional high-CPA vitrification is achieved by using penetrating cryoprotectants (CPAs) such as dimethylsulfoxide (DMSO) and 1,2-propanediol (PROH) at concentrations that are much higher than that used for slow freezing (up to 8 M versus ~1–2 M), which is toxic to cells due to the high CPA-induced metabolic and osmotic (including cell dehydration) injuries. [6] As a result, multiple steps are required to gradually load (before cooling) and unload (after warming) the CPAs to potentially reduce the high-CPA cytotoxicity, which is time-consuming and stressful.…”

“…[6] As a result, multiple steps are required to gradually load (before cooling) and unload (after warming) the CPAs to potentially reduce the high-CPA cytotoxicity, which is time-consuming and stressful. [4b,7] To overcome these problems, low-CPA vitrification has been explored for cell cryopreservation by using various methods and devices such as electron microscopy grid and quartz microcapillary (QMC) [2a,4b,7d,8] to achieve ultra-rapid cooling rates that minimize IIF by reducing the time available for ice nucleation and growth. Although the cooling rates can be very high (up to ~10 6 °C min −1 ) to achieve vitrification (defined as no visible ice formation in this study in accordance with the literature [9] ) by plunging the devices into liquid nitrogen due to the boiling of liquid nitrogen, the warming rate by plunging the devices into an aqueous solution is usually much lower.…”

Improved low-CPA vitrification of mouse oocytes using quartz microcapillary

“…More recently, low-CPA vitrification has been explored for cell cryopreservation by using various methods or devices [8-10,12,16,34,37,39], to achieve ultra-rapid cooling rate that minimizes IIF by reducing the time available for ice nucleation and growth. For example, the miniaturized quartz microcapillary (QMC) has been successfully used to achieve low-CPA vitrification of mouse mesenchymal stem cells (MSCs), mouse embryonic stem cells (ESCs), and mouse oocytes [12,16,39].…”