Assessment of the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model

Abstract

The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. To this end, cryopreservation plays a critical role, serving as an enabling process, providing on-demand access to biological material, facilitating large scale production, storage, and distribution of living materials. Despite serving a critical role and substantial improvements over the last several decades, cryopreservation often remains a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. Studies have illustrated the impact and benefit of controlling cryopreservation-induced delayed-onset cell death (CIDOCD) through various “front end” strategies, such as specialized media, new cryoprotective agents, and molecular control during cryopreservation. While proving highly successful, a substantial level of cell death and loss of cell function remains associated with cryopreservation. Recently, we focused on developing technologies (RevitalICE™) designed to reduce the impact of CIDOCD through buffering the cell stress response during the post-thaw recovery phase in an effort to improve the recovery of previously cryopreserved samples. In this study, we investigated the impact of modulating apoptotic caspase activation, oxidative stress, unfolded protein response, and free radical damage in the initial 24 h post-thaw on overall cell survival. Human hematopoietic progenitor cells in vitro cryopreserved in both traditional extracellular-type and intracellular-type cryopreservation freeze media were utilized as a model cell system to assess impact on survival. Our findings demonstrated that through the modulation of several of these pathways, improvements in cell recovery were obtained, regardless of the freeze media and dimethyl sulfoxide concentration utilized. Specifically, through the use of oxidative stress inhibitors, an average increase of 20% in overall viability was observed. Furthermore, the results demonstrated that by using the post-thaw recovery reagent on samples cryopreserved in intracellular-type media (Unisol™), improvements in overall cell survival approaching 80% of non-frozen controls were attained. While improvements in overall survival were obtained, an assessment on the impact of specific cell subpopulations and functionality remains to be completed. While work remains, these results represent an important step forward in the development of improved cryopreservation processes for use in discovery science, and commercial and clinical settings.

Reenacting Neuroectodermal Exposure of Hematopoietic Progenitors Enables Scalable Production of Cryopreservable iPSC-Derived Human Microglia

“…Importantly, the cellular phenotype as well as the functional immune responses of iPSdMiG are largely maintained after cryopreservation, making our differentiation scheme compatible with future cell banking endeavors. Only basal and induced ROS release levels were increased after thawing, which could point to potential activation of stress response pathways as a result of the cryopreservation process [ 68 ].…”

Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation

“…Post-thaw, cells were allowed to recover for 24 hours to ensure apoptosis could set in, and to avoid over-estimation of recovery associated with short post-thaw incubations. 50,51 Recovery and viability was recorded every 4 hours during this period, determined by trypan blue exclusion, Fig. 1.…”

Biobanks as an important tool in modern translational oncology

“…As a rule, dimethyl sulfoxide (DMSO) is used as an agent preventing cell destruction by water crystals during freezing. However, the use of this approach is not ideal, so alternative methods of cell and tissue cryopreservation that do not use DMSO (Pogozhykh, 2020;, allowing to reduce the amount of DMSO during cryopreservation (Tripathy, Singh, and Das, 2022), and methods minimizing the impact of cryopreservation on cells and reducing cellular stress associated with freezingthawing (Baust, Snyder, Van Buskirk, and Baust, 2022) are developed and applied. Obviously, different cell sub-populations (in human tumor tissue or in microbiota community) may exhibit different sensitivity to preservation (Ryan et al, 2021), thus, during long-term storage and subsequent revitalization of tissues and cells, selective survival of only certain cell sub-populations, or clonal selec-tion, may occur.…”