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

Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells

“…36% using the optimal conditions is not within the acceptable range for reproducibility and scaleup from laboratory R&D to commercial scale processes. Moreover, the reported yields in this study are significantly lower than those reported across various mammalian cell lines in the literature (Eroglu et al 2000, He 2011, Lee et al 2013, Limaye and Kale 2001, Mart ın-Ib añez et al 2012, Serra et al 2011, Wikstr€ om et al 2012, Zhang et al 2010) specifically, 60% post-thawing efficiency has been reported for HEK cells using 5% DMSO dissolved in histidine-tryptophan-ketoglutarate medium (Chaytor et al 2012), which is at least 70% higher than the values obtained for the optimal conditions in 2D using DMSO (Run 8). The latter can be explained by higher viability efficiencies immediately after thawing relative to those observed after 1-7 days culture of the thawed cells (Heng et al 2006).…”

“…In the quiescent state, cells enter an amorphous/glassy phase characterized by low molecular mobility and activity to arrest biophysical and biochemical cellular activities. Successful preservation is governed by complex interactions between the type and concentration of cryo/lyoprotectant cocktails, kinetics of liquid water removal enhanced by 3D conformation, as well as cell-cell and cell-matrix interactions and specific properties of the individual IC cell lines (He 2011, Mart ın-Ib añez et al 2012, Serra et al 2011, Wikstr€ om et al 2012, Zhang et al 2010. The critical variables leading to cryo-injury, use of cryoprotective agents (CPAs) and biophysics of mammalian cell cryopreservation for optimal recovery rates based on modeling of cell dehydration and intracellular ice formation have been extensively reviewed by He (2011) and Mart ın-Ib añez et al (2012).…”

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature?

“…A good, recent example is the CP of human pluripotent (embryonic and induced alike) stem cells (hESc's and iPSC's respectively). Introduction of i) multi-step freezing, ii) ROCK inhibitors in combination with full cell dissociation, and iii) freezing pluripotent SC's in adherent stage as they are prone to anoikis (cell death after cell are detached from extracellular matrix, [Wagh et al, 2011]) have dramatically increased survival and functionality of human pluripotent cells after SF ( [Katkov et al, 2011;Li et al, 2008;MartinIbanez et al, 2009;Mollamohammadi et al, 2009;Stubban et al, 2007;Ware & Baran, 2007], see Chapter by Martin-Ibanez [Martin-Ibanez, 2012] in this Book). It now highly supersedes various vitrification techniques proposed from time to time [Beier et al, 2011;Reubinoff et al, 2001;Zhou et al, 2004] despite what is claimed otherwise by the authors.…”

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

“…An optimal concentration of suitable CPAs should be added to cell suspensions to avoid such injury. Reproduced with permission from Martin-Ibanez et al 84 which have the potential to cause chemical toxicity and osmotic shock to cells. 79 Moreover, vitrification might also lead to potential MSCs contamination with pathogenic agents, due to the direct exposure of the cell suspensions to nonsterile liquid nitrogen.…”

“…Slow freezing involves issues related to a higher risk of freeze injury (e.g., cell death) due to the formation of intra-and extracellular ice during the freezing process. 83,84 To address this issue, optimization of the use of CPA is very important to avoid ice crystal formation by loading the MSCs with the optimum concentration of suitable CPAs (Fig. 1).…”