Characterizing the “sweet spot” for the preservation of a T-cell line using osmolytes

Abstract

This study examined the post-thaw recovery of Jurkat cells cryopreserved in single osmolyte solutions containing sucrose, glycerol or isoleucine, as well as in a combination of the three osmolytes. Cell response was determined using low temperature Raman Spectroscopy and variation in post-thaw recovery with composition was analyzed using statistical modeling. Post-thaw recovery of Jurkat cells in single osmolyte was low. A combination of the osmolytes displayed a non-linear relationship between composition and post-thaw recovery, suggesting that interactions exist between the different solutes. The post-thaw recovery for an optimized multicomponent solution was comparable to that observed using 10% dimethyl sulfoxide and a cooling rate of 1 °C/min. Statistical modeling was used to characterize the importance of each osmolyte in the combination and test for interactions between osmolytes. Higher concentrations of glycerol increase post-thaw recovery and interactions between sucrose and glycerol, as well as sucrose and isoleucine improve post-thaw recovery. Raman images clearly demonstrated that damaging intracellular ice formation was observed more often in the presence of single osmolytes as well as non-optimized multi-component solution compositions.

Characterizing modes of action and interaction for multicomponent osmolyte solutions on Jurkat cells

“…There appears to be a threshold level for the stabilizing effects of the sugars: the post‐thaw recovery increases with an increasing level of sugar and then decreases for increasing level of sugar beyond the threshold level for both SGC and TGC. The outcome is consistent with that observed in our previous work (Pi et al, ).…”

“…Sugar alcohols can also interact with water, and alteration of ice structure by osmolytes has been demonstrated to be influential in cryopreservation outcome (Bailey et al, ). To examine the interactions between osmolytes and water during freezing, Raman images of osmolyte solutions without cells cooled down to −50°C with cooling rate 1°C/min were obtained for SGI353 (sucrose–glycerol–isoleucine; Pi et al, ), SGC353, TGC353, and SMC353 solutions. At this temperature, both ice and unfrozen liquid were present, and distinct differences in the ice morphology were observed with the different solution compositions.…”

“…These compounds are used in nature to stabilize biological systems subjected to environmental extremes (P. Yancey, Clark, Hand, Bowlus, & Somero, 1982). In previous studies, we demonstrated that various osmolytes can act in concert to improve cell viability during cryopreservation (Pi, Yu, Petersen, & Hubel, 2018;Pollock, Yu et al, 2016;Pollock et al, 2017;Pollock, Budenske, McKenna, Dosa, & Hubel, 2016;Yu, Yap, Pollock, & Hubel, 2017). We have shown that certain combinations of sugars, sugar alcohols and amino acids not only maintained post-thaw recovery but also resulted in cells that had improved function compared with those that were cryopreserved with DMSO.…”

Preservation of cell-based immunotherapies for clinical trials

“…These might account for loss of functional cells in cryopreserved T-cell product for immunotherapy. In comparison, DMSO-free freezing media composed of sugars, sugar alcohols and amino acids have been demonstrated to successfully preserve Jurkat cells as a CD3 + CD4 + T-lymphocyte model with high postthaw viability [8,64] and can potentially be used to cryopreserve other types of T cells. Similar to T cells, although NK cells cryopreserved in DMSO have shown significant functional loss in terms of their cytotoxicity [60], a DMSO-free freezing medium composed of poly-l-lysine, dextran and ectoine has demonstrated improved post-thaw cytotoxicity in cryopreserved NK cells [65].…”

“…This restriction complicates the workflow considerably at both the site of manufacture and the site of use. DMSO-free multi-osmolyte solutions have effectively preserved MSCs [7] and T lymphocytes [8] and have provided improved cell stability. Therefore, these CPAs have prolonged the window of manufacturing both prefreeze and post-thaw.…”