Cryopreservation of adherent human embryonic stem cells

Abstract

Standard human embryonic stem (HES) cell cryopreservation methodologies, including slow freezing and vitrification of colonies in suspension, are plagued by poor viability and high differentiation rates upon recovery. To facilitate research studies and clinical applications of HES cells, we have developed a cryopreservation technique based on stabilizing HES colonies adherent to or embedded in a Matrigel matrix. This method increases cell viability by over an order of magnitude compared with cryopreservation in suspension and reduces differentiation. Loading adherent HES cells with the disaccharide trehalose prior to cryopreserving in a dimethylsulfoxide-containing cryoprotectant solution further improves cell viability under certain conditions. Our proposed approach has the potential to reduce the time required to amplify frozen stocks of HES cells, minimize risk of clonal selection during freeze-thaw cycles, and facilitate storage of HES cell clone libraries.

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

“…Finally, trehalose addition to the cryopreservation medium containing DMSO and fetal bovine serum (FBS) has been proven to increase the viability of hematopoietic precursor cells from 7% to 20% and improved membrane integrity in cryopreserved fetal skin cells (Erdag et al, 2002;Zhang et al, 2003). Ji et al showed that trehalose loading into adherent colonies of hESCs prior to cryopreservation results in small, but significant improvements in cell viability when combined with DMSO treatment and high FBS concentrations (Ji et al, 2004). In the same line of results, it has been demonstrated that trehalose addition to the freezing and post-thawing medium of hESC colonies cryopreserved in suspension in freezing medium containing 10% DMSO, increased the recovery rate by ~3 folds (from 15 to 48%) (Wu et al, 2005).…”

“…Similarly, hepatocytes sandwiched between two layers of collagen provide enhanced viability and protein secretion compared with cells preserved in solution (Birraux et al, 2002;Koebe et al, 1990;Koebe et al, 1999). Taking these results as a proof of principle, hESCs were successfully cryopreserved as adherent colonies in 24 well plates in medium containing 10% DMSO + 30% FBS by Ji et al (Ji et al, 2004). This approach demonstrated that hESCs frozen as adherent colonies were five times more viable than clumps of colonies frozen in suspension.…”

“…This is a decisive process for the survival of hPSC colonies frozen in suspension that is rarely achieved due to the massive cell death or cell damage experienced within the colony during cryopreservation. Second, the maintenance of a continuous extracellular matrix signaling may also play a role in the enhanced viability and reduced differentiation of hESCs cryopreserved in an adherent state (Ji et al, 2004). The disadvantage of this technique is that large scale storage is not feasible because hPSCs attached to plates cannot be stored at high density.…”

Cryopreservation of Rat Bone Marrow Derived Mesenchymal Stem Cells by Two Conventional and Open-Pulled Straw Vitrification Methods

“…Therefore, this difference in recovery may be due to differences in cell lines, freezing and thawing protocols, or growth substrate (49).…”

Is stem cell chromosomes stability affected by cryopreservation conditions?

“…Trounson's group demonstrated that current methods of freezing, storage and recovery trigger apoptosis and spontaneous differentiation of hES with consequent loss of pluripotency Reubinoff et al 2000). Several other groups have also confirmed very low survival of hES cell after slow freezing with DMSO (Heng et al 2005;Ji et al 2004;Kim et al 2004).…”