T cells, which are crucial in cancer therapy, activate immune functions upon pathogen invasion (1–3), and the immune function will be exercised by the release of tumor necrosis factor alpha (TNF-α), interferon gamma (INF-γ), interleukin 2 (IL-2), and other cytokines (4, 5). The surge in the use of immune cells, such as the production of chimeric antigen receptor (CAR) T cells (6, 7), has underscored the importance of cryopreservation, a challenge that still plagues T-cell immunotherapy (8–12). Cryopreserved immune cells find widespread use in adoptive transfer, prompting a need for efficient cryopreservation methods (13, 14). Despite notable successes in multiple practical applications, cryopreservation still introduces challenges such as impaired cytotoxicity, altered gene expression, and changes in the T-cell phenotype (15–17). It remains crucial to enhance the efficacy of cryopreservation for clinical purposes (6, 18–20).

Moreover, the rising demand for allogeneic T-cell clinical transplantation has intensified the need for improved storage technologies (21) in order to reduce the costs of equipment and labor and to mitigate risks during transportation (22, 23). Presently, due to the thermodynamic instability of conventional cryoprotective solutions that solely rely on the use of small-molecule cryoprotectants such as dimethyl sulfoxide (DMSO) at −80°C, clinical studies predominantly rely on storage in cryogenic liquid nitrogen (LN2) facilities that operate at a temperature range between −196°C (in the liquid phase) and approximately −120°C (in the gas phase). However, the use of LN2 presents logistical and operational challenges. Addressing the core issue of the stability of the medium at −80°C becomes pivotal for effective cryopreservation (24, 25). Considering the vulnerability of T cells to the damaging effects involved in cryopreservation procedures, maintaining both cell vitality and functionality after long-term storage at −80°C is technically challenging for conventional cryopreservation media. This difficulty primarily arises because conventional cryopreservation solutions—which typically contain small-molecule cell-permeating cryoprotectants such as DMSO and ethylene glycol, as well as macromolecular cryoprotectants such as fetal bovine serum (FBS) components and various linear or branched synthetic polymers such as polyethylene glycol and dextran—undergo ice recrystallization (26–28). This process spontaneously occurs at temperatures higher than those in LN2 storage, which leads to the gradual formation of enlarged ice crystals. These crystals, growing from smaller ones formed during the initial freezing, inflict severe mechanical damage on regular mammalian cell types. Numerous mammalian cell types cryopreserved in conventional media have shown considerable viability loss after short-term storage at −80°C, with complete viability loss over longer periods (e.g., 1 year) (29–32).

No systematic research has been conducted to comprehensively evaluate the impact of storage at −80°C on the post-thaw viability, the proliferation efficiency, and, more importantly, the cytotoxicity of primary T cells. In this work, we aimed to not only address these research gaps but also demonstrate a promising solution to provide a new direction in immune cell cryopreservation and transportation that can overcome the technical challenges in supply chain management for immunotherapies based on the use of T cells and other immune cells. In a word, this study introduces a novel polymer-based cryopreservation medium uniquely designed to enhance the outcome of the cryo-storage of T cells at −80°C by preventing the recrystallization process and its associated damaging effects.

Efficient long-term cryo-storage of regular mammalian cell types at −80°C relies on the efficacy in preventing ice recrystallization and its damaging effects. Our thermal and molecular dynamic studies have revealed that Ficoll molecules, a polysaccharide forming a unique, highly compact spherical shape, can completely prevent recrystallization and its damaging effects (25, 37, 38). In models using human embryonic and induced pluripotent stem cells, Ficoll-based cryopreservation at −80°C yielded post-thaw viability and proliferation efficiency comparable to that of the traditional use of FBS+DMSO and LN2 storage over 1 year. We also performed a viability comparison assay on the human T cells in C80EZ^® cryopreserved at −80°C and the T cells in FBS cryopreserved in LN2 for 2 months ( Supplementary Figure S1A ). The results showed that the T cells cryopreserved in these two conditions have comparable post-thaw viability. Our resulting commercial product, the C80EZ^® medium, also incorporates a low concentration of chondroitin sulfate as an anti-apoptotic agent to mitigate cell death from biophysical impacts such as intracellular water loss during freezing. C80EZ^® has been beneficial in various industrial applications, eliminating the need for serum in cell cryopreservation, reducing the DMSO concentration, and, most notably, removing the dependence on LN2 storage. Its effectiveness in preserving engineered insect cells over long-term storage at −80°C has also been confirmed (36).

This study aimed to highlight the superiority of C80EZ^® by comparing it with high-concentration FBS-based methods. Although commercial media such as CryoStor^® CS10 are used clinically, especially for T-cell cryopreservation, their post-thaw viability and functionality fall interior in comparison to the use of high FBS concentrations (e.g., 95%); moreover, they are not recommended for long-term −80°C storage (39). The primary advantage of CS10 is the exclusion of animal serum, addressing regulatory concerns. Hence, our study will focus on comparing C80EZ^® with an FBS+DMSO control.

There have also been several studies into novel cryoprotectants of T cells that have shown exciting results in terms of the high viability and the cytotoxicity of post-thaw T cells (40, 41), but they have all focused on reducing the cytotoxicity of DMSO or replacing FBS in the cryopreservation medium. On the other hand, our study was dedicated to replacing LN2 with higher temperatures, which can be achieved with general equipment.

In our control group using an FBS-based medium, similar to previous studies on the storage of T cells in LN2, the cell proliferation and toxicity were affected (42, 43). However, the effects observed in our control group were more pronounced than those seen with LN2 storage. This difference could be attributed to cellular damage associated with the recrystallization process in −80°C freezers, a phenomenon also noted in other cell types stored at −80°C (28). Conversely, the results from our study revealed similar viability of the T cells stored in C80EZ^® at −80°C to those cryopreserved in LN2, indicating that C80EZ^® significantly improved the protection ability during the cryopreservation, similar to its effects on engineered insect cells or stem cells (25, 36).

The examination of T-cell functionality, encompassing cytotoxicity, stemness, differentiation, and TNF-α and IFN-γ release after cryopreservation at −80°C, solidifies the superiority of C80EZ^®. The proportion of naive T cells, the percentage of CD8⁺ T cells, and the release of TNF-α and IFN-γ were notably higher in the C80EZ^® group compared with that in other conditions. Moreover, C80EZ^® demonstrated the capability to maintain the proportion of CD8⁺ effector T cells within the total viable T-cell population after cryo-storage at −80°C. These findings collectively substantiate the adoption of C80EZ^® as a potentially superior choice for biobanking immune cells. Ongoing studies are in progress to confirm the efficacy of longer-term storage.

However, although we have performed a series of studies on the biological parameters, the current study is limited to the 2-month storage period. Based on our research with stem cells, the reduction in post-thaw viability would likely be significantly more pronounced if a longer storage period were studied. No viability would logically result in null proliferation and functionality. Long-term storage will be evaluated to substantiate our prediction, as a future direction. Moreover, through our proliferation results, despite the use of the C80EZ^® medium, post-thaw proliferation remains suboptimal, indicating that this issue may not be resolved solely by enhancing the cryopreservation medium. Instead, it could potentially be addressed by refining the post-thaw culturing protocols, such as the inclusion of Rho-associated protein kinase (ROCK) inhibitors (44) or other agents or the improvement of the culture conditions.

In conclusion, our study provides valuable insights into overcoming challenges in T-cell cryopreservation, introducing C80EZ^® as an innovative solution to enhance T-cell viability, proliferation, and functionality. This holds significant promise for advancing the efficacy and accessibility of T-cell-based immunotherapies, as the technology addresses prevalent practical and technical challenges in quality control and supply chain management associated with the therapeutic process.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human blood cells were reviewed and approved by the American Red Cross Institutional Review Board (IRB).

WZ: Investigation, Methodology, Writing – original draft. CW: Investigation, Writing – review & editing. YS: Methodology, Writing – review & editing. YP: Methodology, Writing – review & editing. XM: Methodology, Writing – review & editing. XK: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. XH: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.