Freezing Medium Containing 5% DMSO Enhances the Cell Viability and Recovery Rate After Cryopreservation of Regulatory T Cell Products ex vivo and in vivo

Abstract

Cell therapies have significant therapeutic potential in diverse fields including regenerative medicine, transplantation tolerance, and autoimmunity. Within these fields, regulatory T cells (Treg) have been deployed to ameliorate aberrant immune responses with great success. However, translation of the cryopreservation strategies employed for other cell therapy products, such as effector T cell therapies, to Treg therapies has been challenging. The lack of an optimized cryopreservation strategy for Treg products presents a substantial obstacle to their broader application, particularly as administration of fresh cells limits the window available for sterility and functional assessment. In this study, we aimed to develop an optimized cryopreservation strategy for our CD4+CD25+Foxp3+ Treg clinical product. We investigate the effect of synthetic or organic cryoprotectants including different concentrations of DMSO on Treg recovery, viability, phenotype, cytokine production, suppressive capacity, and in vivo survival following GMP-compliant manufacture. We additionally assess the effect of adding the extracellular cryoprotectant polyethylene glycol (PEG), or priming cellular expression of heat shock proteins as strategies to improve viability. We find that cryopreservation in serum-free freezing medium supplemented with 10% human serum albumin and 5% DMSO facilitates improved Treg recovery and functionality and supports a reduced DMSO concentration in Treg cryopreservation protocols. This strategy may be easily incorporated into clinical manufacture protocols for future studies.

Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research

“…In contrast, monocytes better withstood the procedure, as there was a relative overrepresentation of monocytes compared to all CD45+ cells after cryopreservation compared to the distribution before freezing. The unequal recovery rates between monocytes and T cells could be due to different susceptibilities of the individual leukocyte populations to the used cryoprotectants, as, for example, increasing dosages of DMSO led to significantly reduced recovery of Treg cells [39]. Interestingly, this increase was not evenly dispersed over all monocyte subsets, as the relative frequency of "younger" classical monocytes was increased and the amount of "aged" non-classical monocytes [40] was significantly reduced after cryopreservation.…”

Untangling the Knots of Regulatory T Cell Therapy in Solid Organ Transplantation

“…Widening the window between the collection and application of Tregs for adoptive therapy would increase the flexibility of their clinical use. Because preservation techniques can potentially change the yield, viability, and activity of Tregs, they are considered a new therapeutic biological product from a regulatory point of view ( 55 ).…”

“…Tregs can be cryopreserved before isolation (as peripheral blood mononuclear cells, PBMCs), just after isolation, or after the expansion phase. Treg cell recovery rates from cryopreserved PBMCs fluctuate between 35 to 63% ( 55 – 61 ). Using isolated Tregs cryopreserved in liquid nitrogen for up to one year, Peters et al.…”

“…Kaiser et al. found better recovery rates and cellular viability by using cryogenic solution of 5% DMSO instead of 10% DMSO ( 55 ). Cryopreserved Tregs after three or four cycles of re-stimulation did not alter their original phenotype or suppressive function ( 56 ).…”

Tregs in Autoimmunity: Insights Into Intrinsic Brake Mechanism Driving Pathogenesis and Immune Homeostasis

“…Human Treg biology has been a challenge due to their relatively low frequency in peripheral blood, lower proliferation rates and loss of function, survival and phenotypic stability in long-term cultures and after freeze thaw (90)(91)(92). Recently, many of these challenges have been overcome, opening the field of human Treg biology to be used in adoptive cell therapy, high-through put screens and novel genome editing techniques like CRISPR (clustered, regularly interspaced, short palindromic repeats)/ Cas9 (CRISPR associated protein 9) (93)(94)(95). These advances on top of the decades of fundamental research support the use of Tregs in adoptive cell therapy.…”