C5 - Developing new cryopreservation strategies and formulations for cell therapy applications

Introduction: : Cryopreservation is an essential process that potentiates successful implementation of cell-based therapies— preventing ice crystal formation, regulating osmotic stress, and maintaining integral cell function. CAR T-cell and stem cell therapies have significantly advanced treatment of hematological malignancies and chronic illnesses. Despite these successes, cell-based therapies continue to face several challenges that limit broad translation, including inconsistent post-thaw viability. Suboptimal cryopreservation strategies can result in cell death or loss of therapeutic potency.
Dimethyl sulfoxide (DMSO) supplemented with serum has long been the preferable cryoprotectant formulation; however, high DMSO concentrations can interfere with enzymatic activity and cell proliferation, while serum introduces batch variability and potential toxicity. It is therefore essential to establish new cryochain methods and strategies that mitigate variability and downstream complications. We developed a DMSO-reduced and serum-free formulation using a new two-pronged cryopreservation approach whereby protectant agents are not only supplemented extracellularly but also delivered intracellularly via electroporation. Cryopreservation strategy translatability was evaluated using human and murine-derived CAR T-cells that target hematological or solid tumors, and multipotent stem cells.
Our results demonstrated significantly improved post-thaw viability, proliferation, functionality, and in vivo efficacy compared to conventional methods. Further, high-content omics data indicated that our strategy was more effective in mediating protein and metabolites responsible for cell growth and proliferation than conventional DMSO-serum based approaches. These findings support a next-generation cryopreservation approach that enhances cell quality and therapeutic consistency across heterogenous cell types. This strategy can improve biobanking scalability and reliability, along with expanding the clinical impact of advanced cell-based therapies.

Materials and

Methods: : Cell Lines: Human anti-CD19, murine anti-EGFR-vIII CAR T- and human mesenchymal stem cells (MSCs) were used to assess cryochain efficacy. Raji-luciferase and EGFRvIII-CT-2A were used as target tumor cell lines to evaluate CAR-T cell potency.
Cryopreservation: Cells were resuspended with trehalose or sucrose, and subsequently electroporated at varying voltage and capacitance, and waveforms to induce intracellular cryoprotectant agents. Cells were pelleted and then mixed with sugar-supplemented DMSO formulations. Samples were cooled and frozen to -80C overnight at -1C/min and then maintained in cryogenic storage systems. Our approach was compared to conventional cryopreservation strategies that supplement DMSO-serum formulations extracellularly for downstream experiments.
In vitro post-thaw assessment: Cells were thawed and maintained for 5 days to examine immediate and long-term viability and expansion using PrestoBlue. Differences in surface marker expression characteristics were determined using flow cytometry. To evaluate preservation of cell functionality and potency, CAR T-cells were co-incubated with Raji-luciferase or EGFRvIII-CT-2A cells overnight. Co-culture supernatant was additionally isolated to measure IFN-gamma and granzyme B secretion using ELISAs. CAR T-cell potency was correlated to luciferase activity and cytotoxic granule levels. To evaluate preservation of MSC multipotency, thawed MSCs were treated with osteogenic differentiation media and subsequently probed for osteogenic markers using RT-PCR.
Anti-CD19 CAR T-cell efficacy in vivo: NSG mice were injected with 5x105 FLuc+ Raji cells to establish lymphoma models. Mice were then treated with 1x106 CAR T-cells or untransduced T-cells three days after Raji injection. Lymphoma progression was monitored every four days for seven weeks via bioluminescent signal via IVIS.

Results, Conclusions, and Discussions:: Electroporation-based cryopreservation approach and formulation significantly improved post-thaw expansion and viability compared to conventional freezing strategies. Using high-content omics, we found that our approach preserved proteins and metabolites associated to ribosome biogenesis and translational machinery —pathways directly linked to cell growth and proliferation— more effectively than DMSO-serum methods. CAR T-cells cryopreserved using our method were more potent against Burkitt’s lymphoma cells. These cells enhanced cytotoxic granule secretion that induced tumor cell cytotoxicity. Further, lymphoma-bearing mice treated with these CAR T-cells showed a significant survival advantage compared to those treated with conventionally preserved cells. MSCs cryopreserved using electroporation-based methods significantly increased post-thaw expansion, retained multipotency, and exhibited osteogenic phenotypes after differentiation treatment. We are convinced that we present a more robust freezing strategy and formulation that preserves the integrity, potency, and functionality of thawed cells compared to conventional methods.
There have been immense strides in the cryopreservation landscape with leveraging different cryoprotectants that highlight the importance of long-term cell biobanking and the need to develop alternative freezing strategies that can simplify formulations and reduce toxic cryoprotectant agents. For example, CAR T-cell therapy efficacy against tumors is dependent on the number of injected viable cells. Clinical outcomes demonstrated that patients receiving higher doses of viable CAR T-cells had 86% one-year survival, compared to 59% for lower doses. Yet, developing a new formulation that simultaneously achieves consistent post-thaw viability and maintains functionality is challenging. We incorporated two ubiquitous sugars (trehalose or sucrose) used for stabilization that enabled DMSO reduction and serum-free formulations. Electroporation was used to intracellularly deliver impermeable sugars. We assessed method applicability in a diverse range of cell types and species, which showed consistent improvements in post-thaw performance. Although electroporation is one of the several ways to incorporate impermeable biologics, we demonstrated that replacing serum and high DMSO concentrations with sugars enables immediate-use formulations compatible with scalable manufacturing. This workflow has broad translational potential across cell-based therapies and contributes to a better understanding of how cryoprotectant composition impacts cell fate and therapeutic efficacy.

Acknowledgements and/or References (Optional):: This work was supported by the Ludwig Center at MIT Postdoctoral Fellowship