A Novel Multicomponent DMSO Free Cryococktail and Recovery Reagent for the Improved Cryopreservation of NK Cells

Abstract The ultimate goal of cryopreservation (CP) is to develop a biobanking process that results in a post-thaw level of cell viability and function similar to a sister population that has not undergone CP. Most methods used for the clinical level CP of human Natural Killer (NK) cells and other cell therapy cells result in poor recovery due to post-thaw necrosis and apoptosis yielding an inferior cell product which is infused into patients. Our research team has shown through dozens of publications that human stem and somatic cells undergo delayed onset cell death triggered primarily by apoptosis that occurs over a several day period post thaw. These publications support the following questions critical to the clinical use of CP solutions: (1) How much of the observed in vivo cell loss associated with post-thaw infusion of cryopreserved cells is a result of the manifestation of delayed onset cell death that is attributable to the CP solution and post-thaw protocol used in the clinic? (2) What triggers the apoptotic cascade that results in delayed onset cell death? One hypothesis to be tested in this Phase I proposal is that this delayed onset cell death may be a result of CP-triggered premature cell aging and senescence – a hypothesis that is supported by our group’s publications showing that resveratrol, an activator of the Sirtuin (longevity) genes, can improve post hypothermic preservation outcome by up to 10 fold in human hepatocytes and mesenchymal stem cells. But this agent could also be acting through other molecular means as well. Given, however, a July, 2023, publication reporting that select chemical cocktails have been formulated that can reverse the cell aging process this hypothesis can now be tested using CP solutions and post-thaw revival formulations that contain these non-proprietary age-reversing chemicals. Since, over half or more of all cell therapy products in clinical trials today, including NK cells, are cryopreserved in 10% DMSO which is highly toxic, many studies have investigated ways to improve CP as well as to use alternative cryoprotective agents (CPAs) such as sugar alcohols to avoid the known toxicity of DMSO. Indeed, a symposium at Cryo2023 was solely dedicated to discussing alternatives to DMSO as CPAs making this focus a high priority in the CP sciences. Yet, to date no new CP process that eliminates DMSO has been developed commercially. To achieve this milestone, first, DMSO must be replaced with CPAs that can prevent the formation and re-crystallization of lethal ice during the CP process. Second, a post-thaw recovery conditioning dilution/infusion reagent must be developed that can prevent post-thaw delayed onset cell death and improve NK cell viability and function immediately following thawing. Specific Aims of this project are the following: SA1) Develop Chillout - a non-toxic CPA cryococktail to replace DMSO; SA2) Formulate RevitalICE – a first in class CP dilution reagent designed to ameliorate post-thaw apoptosis/necrosis increasing NK survival and infusion efficacy and SA3) Evaluate NK cell cytotoxic function following CP. Hypotheses to be tested in this Phase I project are the following: (1) The apoptosis-dependent delayed onset cell death is initially triggered by CP-induced premature cell aging leading to poor CP outcome; (2) NK CP cell survival and function can be improved through the addition of cell stress modulators and age-reversing chemical cocktails; and (3) elimination of DMSO will further reduce NK stress associated with CP. Hypotheses to be tested in the Phase II project are the following: (1) A DMSO free CP solution can be developed with cell stress and age-reversing modulators that is FDA and clinically acceptable for therapeutic application and results in better post CP outcome than traditional solutions containing 10% DMSO: (2) Delayed onset cell death occurs in vivo in animal models if NK cells are stored in DMSO-containing CP solutions but does not occur if the proposed Chillout and RevitalICE containing cell stress and age-reversing modulators are used as the CP formulations and post-thaw revival solutions. These efforts will result in improved NK and other cell therapy cell CP and thereby improve clinical outcome for patients receiving these cells.