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The ROCK inhibitor, Y-27632, protects stem cells from dissociation-associated apoptosis during cryopreservation and increases cell viability post-thawing. This blog post discusses why the ROCK inhibitor is required, provides evidence for its use with different stem cell types, and outlines how the ROCK inhibitor protects cells during cryopreservation.
Stem cells represent a theoretically unlimited supply of differentiated cells for use in clinical cell therapies, as well as for toxicology studies and drug discovery. The appropriate storage of stem cells is important in order to provide an accessible source of cells for use. Stem cells are stored as frozen samples, following cryopreservation. Methods of cryopreservation include fast or slow freezing with cryoprotectants, such as DMSO. Fast freezing methods, also called vitrification are labor intensive and are reported to be unsuited for the bulk quantities of stem cells required for clinical application of cell therapies. Slow-freezing methods are also not efficient for stem cell cryopreservation, showing around 10% survival rates.
A major breakthrough in stem cell cryopreservation came in 2007, when Watanabe et al. showed that a selective p160-Rho kinase (ROCK) inhibitor (Y-27632, Cat. No. 1254) can improve cell viability during cryopreservation. Human embryonic stem cells (hESCs) are prone to apoptosis on cell detachment and dissociation in the first stage of stem cell culture. To inhibit dissociation-associated apoptosis, Watanabe et al. tested a range of caspase inhibitors, growth factors, trophic factors and kinase inhibitors for their effect on hESC viability. They determined that the ROCK inhibitor diminished dissociation-associated apoptosis and increased cloning efficiency from approximately 1% to 27%. The protective abilities of the ROCK inhibitor also enabled serum-free suspension (SFEB)-cultured hESCs to survive and differentiate into cortical and basal telencephalic progenitor cells.
Subsequently, Li et al. (2009) showed that Y-27632 improves the survival of single hESCs after cryopreservation. When added to the post-thaw culture medium for 24h, the ROCK inhibitor increased the number of cells that remained attached following freeze-thawing showing that this compound increased adherent properties of hESCs and protected against apoptosis. Cells retained characteristic morphology, karyotype and pluripotency following cryopreservation and long-term culture.
The ROCK inhibitor has also been investigated in cryopreservation of human bone marrow-derived mesenchymal stem cells (MSCs). When added to post-thaw culture medium, the ROCK inhibitor increased the proportion of adherent, viable cells. Similarly, the ROCK inhibitor improves survival of human pluripotent stem cells (hPSCs) when included in cryopreservation medium and post-thaw culture medium (figure 1).
Figure 1: An example protocol for cryopreservation and thawing of hPSCs, incorporating the ROCK inhibitor Y-27632.
Exactly how Y-27632 protects cells from apoptosis and increases adherence can be deduced from the physiological roles of ROCK. This enzyme is involved in a range of biological processes including cell adhesion, proliferation, differentiation and apoptosis, depending on the cell type under investigation. ROCK inhibitors are thought to counteract apoptosis by enhancing cell-cell adhesion via modulation of gap junctions, increasing adhesive properties and enhancing post-dissociation cell aggregation.
Heng (2009) Effect of Rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow derived mesenchymal stem cells. Tissue Cell. 41, 376. PMID: 19261317
Li et al (2009) ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod. 24, 580. PMID: 19056770
Liu & Chen (2014) Cryopreservation of human pluripotent stem cells in defined medium. Curr Protoc Stem Cell Biol. 31, 1.
Watanabe et al (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 25, 681.