das-et-al-2024-human-natural-killer-cells-cryopreserved-without-dmso-sustain-robust-effector-responses

1. Introduction to Natural Killer (NK) Cells

  • Overview of NK cells:

    • Innate ability to eliminate targets without prior antigen sensitization.

    • Not significantly associated with graft-versus-host disease (GvHD).

    • Considered allogeneic due to ability to target mismatched MHC settings.

  • Significance in immunotherapy:

    • Safety and functional effectiveness make them attractive for allogeneic therapies.

2. Cryopreservation of NK Cells

  • Importance and necessity:

    • Supporting logistics for decentralized therapy product deployment.

    • Cryopreservation is essential for transportation and storage from manufacturing to clinical sites.

  • DMSO as a Cryoprotective Agent (CPA):

    • Most commonly used due to its ability to penetrate cell membranes and reduce ice formation.

    • Associated with potential toxicity and impacts on NK cell function, including epigenetic changes.

  • Need for alternatives to DMSO:

    • Alternatives are sought to avoid the disadvantages linked to DMSO, particularly in maintaining cell function post-thaw.

3. Study Objectives

  • Main Investigations:

    • Explore the feasibility of using non-DMSO CPAs for cryopreservation of human NK cells.

    • Assess viability and functionality of NK cells post-thaw.

4. Methods

4.1 NK Cell Isolation and Culture

  • Isolation: From fresh whole blood of healthy volunteers using negative immunomagnetic selection.

  • Culture Conditions: Maintained in RPMIf medium, supplemented with cytokines (IL-2, IL-21).

4.2 Cryopreservation Techniques

  • Cells treated with varying concenttations of DMSO and other CPAs (e.g., ethylene glycol, propylene glycol).

  • Sample parameters for cryopreservation and thawing detailed.

4.3 Viability Assessment

  • Viability Determination: Calculated using Trypan blue exclusion method pre- and post-thaw.

4.4 Cytotoxicity Assays

  • Evaluated NK cell killing capacity against GBM43 tumor cells via co-culturing at varying E/T ratios.

4.5 Flow Cytometry for Functional Analysis

  • Evaluating expression of activation markers (CD107a, IFN-γ) and NK cell functionality post-thaw.

5. Results

5.1 Viability of NK Cells

  • DMSO vs. Non-DMSO CPAs: - DMSO preserved higher viability compared to several non-DMSO formulations.

    • Ethylene glycol (EG) showed comparable efficacy to DMSO post-thaw.

5.2 Functional Assays

  • Cytotoxicity Results:

    • NK cells cryopreserved with DMSO demonstrated effective cytotoxicity against GBM cells.

    • EG-cryopreserved NK cells also showed comparable cytotoxicity.

5.3 Characterization of Thawed NK Cells

  • Degranulation and Cytokine Production:

    • EG-cryopreserved NK cells equivalently demonstrated degranulation (CD107a expression) and IFN-γ production compared to DMSO.

5.4 Migration Assays

  • Impacted Migration: Regardless of CPA used, both DMSO and EG thawed NK cells showed reduced migration capacity compared to fresh NK cells.

6. Discussion

  • Impact of Cryopreservation: Highlighted importance of selecting CPAs that facilitate cellular viability while minimizing toxicity.

  • Significant findings of EG: Showed strong potential as an alternative CPA, maintaining NK cell viability and functional characteristics post-thaw.

  • Future Directions:

    • Emphasis on exploring clinical implications and safety of EG for NK cell cryopreservation in immunotherapy contexts.

7. Conclusion

  • The study presents a promising example of the use of non-DMSO CPAs for NK cell cryopreservation, potentially facilitating safer and more effective NK cell-based immunotherapies.