Storage Temperature Stability of Therapeutic Cell Types

Dec 15, 2025

Learn how storage temperature affects CAR-T, stem cells, MSCs, and iPSCs, including cryogenic thresholds, excursions, and quality risks.

Abstract

Therapeutic cells are "living drugs," and their viability and potency can change quickly if storage temperatures drift outside validated ranges. For most cell therapies, the safest long-term strategy is true cryogenic storage-kept below the glass transition (approximately –135°C for aqueous systems) to minimize damaging events like devitrification and ice recrystallization.[1][2][3][4]

Temperature Stability Thresholds

Storage Condition Temperature Range Clinical Application
Cryogenic Storage ≤ –150°C Long-term stability for most cryopreserved therapeutic cells. Minimizes ice recrystallization.
Glass Transition ~–135°C Critical threshold where ice structure changes become possible. Above this, devitrification risk increases.[1][2][3][4]
Ultra-Low Freezer –80°C Not true cryogenic. Limited duration only; gradual viability loss without specific validation.[5]
Refrigerated 2–8°C Short-term hold. Metabolism slows but continues. Typically hours to 1–3 days depending on cell type.
Ambient 20–25°C Short window only. Cell stress accelerates. Generally hours-scale for most therapies.

1. CAR-T Cells

Chimeric Antigen Receptor T-Cell Products

Storage Requirements (Long-Term)

Commercial CAR-T products are typically labeled for storage in vapor-phase liquid nitrogen at very low temperatures (commonly ≤ –150°C, sometimes ≤ –120°C depending on product).[6][7][8][9][10][11]

  • YESCARTA:Store frozen in LN₂ vapor phase ≤ –150°C[10]
  • TECARTUS:Store frozen in LN₂ vapor phase ≤ –150°C (label describes single temporary –80°C hold option under defined conditions)[11]
  • KYMRIAH:Stored/shipped in LN₂ vapor phase (SBRA describes ≤ –120°C)[8][9]

Temperature Excursion Thresholds

Above ~–135°C: Crossing the glass transition threshold enables damaging ice structure changes.[1][2]

Dry ice (–78°C): Far warmer than cryogenic; limited transfer scenarios only if validated. Not equivalent to LN₂ storage.[5]

2–8°C (Fresh/Non-Cryopreserved): Short shelf-life requiring stability data support:[6][12]

  • Brezinger-Dayan et al.: stability measured at 4 hours at 5 ± 3°C[13]
  • Petrella et al.: critical quality attributes stable during 24 hours at 4°C[14]
  • Luanpitpong et al.: products stable at 4°C for at least 24 hours[15]
Quality Degradation Indicators

Reduced post-thaw viability; shifts in phenotype or activation state; potency drift (killing capacity, cytokine profiles); increased lot-to-lot variability after similar handling.

Operational Requirements

CAR-T requires validated cold chain behavior: defined excursion limits, continuous monitoring, documented investigations when limits are exceeded.

2. Hematopoietic Stem/Progenitor Cells

HSC/HPC Products

Storage Requirements

Long-Term (Cryopreserved): ≤ –150°C for banking, retention, and consistent post-thaw performance. FDA guidance addresses validated processes and ongoing quality control.[17]

Short-Term (Fresh Products):

  • Refrigerated (2–8°C): Commonly used for ~48 hours, sometimes up to ~72 hours with validation
  • Tran et al. (2024): refrigeration at 4°C better preserves attributes vs. room temperature[18]
  • Jansen et al.: viable CD34+ cells retained during 4°C storage[19]
  • Antonenas et al.: controlled cold storage reduces deterioration[20]
Quality Degradation Indicators

Lower viable CD34⁺ recovery; reduced colony-forming units (CFU); declining engraftment performance with prolonged non-ideal holds.

Operational Strategy

Clear split approach: (1) Cryogenic for banked or delayed products; (2) Refrigerated workflow only when infusion timing is predictable and tightly managed.

3. Mesenchymal Stromal Cells

MSC Products

Storage Requirements

Long-Term (Cryopreserved): ≤ –150°C for consistency and banking

Short-Term Holds: Solution-dependent stability profile

  • 4°C / 2–8°C: MSC survival varies 1–2 days in appropriate media; can collapse rapidly in simple saline-like buffers
  • Nofianti et al.: adipose-derived MSC viability differed substantially between culture medium vs. normal saline, demonstrating formulation-dependent stability[21]
Critical Factor

Post-thaw time in cryoprotectant (DMSO) can be the limiting factor even when temperature is controlled. Many workflows require rapid dilution/removal and quick administration.

Quality Degradation Indicators

Delayed apoptosis after thaw; functional potency drift (immunomodulation, differentiation potential); phenotypic changes from stress and rewarming.

4. Induced Pluripotent Stem Cells

iPSC Products

Storage Requirements

Cryopreserved: ≤ –150°C for banks and long-term stability. LN₂ storage is the default because conventional cryoprotectant systems can be thermally unstable at –80°C:[22][23]

  • Yuan et al. (2016): long-term storage requires LN₂; –80°C enables ice recrystallization and progressive viability loss[22]
  • –80°C storage considered short interim step only, not long-term unless validated
  • 2–8°C refrigerated shelf life not standard practice
  • Post-thaw: require rapid recovery into controlled culture conditions
Quality Degradation Indicators

Poor recovery/attachment after thaw; apoptosis and loss of colony-forming efficiency; drift in pluripotency markers and genomic stability.

Operational Focus

Bank integrity: redundancy, documentation, and strict cryogenic continuity.

Regulatory Compliance Requirements

Quality expectations across CAR-T, HPC, MSC, and iPSC programs consistently include:

  • Defined storage temperature ranges with validated stability claims
  • Continuous monitoring (freezers, LN₂ systems, shippers)
  • Documented excursion management: event details, duration, maximum temperature, impact assessment, disposition decision[7]
  • Traceability and chain-of-custody for patient-specific products
  • Release/lot acceptance criteria including viability and potency. FDA guidance: minimum acceptable viability specification generally 70% for somatic cellular therapies[12]

Summary: Temperature Control Ladder

Temperature Stability Profile Application
≤ –150°C Best long-term stability CAR-T, HPC, MSC, iPSC (cryopreserved)
~–135°C Glass transition threshold Above this: increased risk
–80°C Limited/validated only Not "forever storage"
2–8°C Short hold window Hours to ~1–3 days (cell type dependent)
20–25°C Shortest window Hours-scale for most therapies
Clinical Conclusion

Temperature excursions alter living product integrity. Modern biostorage requires validated cold chain execution: redundant systems, continuous monitoring, documented excursion response, and quality documentation meeting regulatory inspection standards.

References

  1. Dobruskin L. Glass Transition in Cryopreservation. PMC. 2024.
  2. Glass Transition Temperature and Recrystallization Risk. Azenta Life Sciences. 2023.
  3. Cryopreservation Guide: Transfer from –80°C to Below –135°C. Sartorius.
  4. Shah N. Cold Chain in Cell Therapy: 2–8°C Fresh vs < –150°C Cryo. PMC. 2023.
  5. Temperature Stability Considerations. Nature.com.
  6. FDA. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products. U.S. Food and Drug Administration.
  7. FDA. 21 CFR 1271.260 Storage Controls and Temperature Monitoring. eCFR.
  8. FDA. KYMRIAH Package Insert and Medication Guide. U.S. Food and Drug Administration.
  9. FDA. Summary Basis for Regulatory Action (KYMRIAH). U.S. Food and Drug Administration.
  10. YESCARTA Prescribing Information: Storage ≤ –150°C, LN₂ Shipper. Mayo Clinic.
  11. FDA. TECARTUS Package Insert: LN₂ Storage; Defined –80°C Single-Hold Allowance. U.S. Food and Drug Administration.
  12. FDA. Guidance for FDA Reviewers and Sponsors: Viability Spec ~70% General Minimum. U.S. Food and Drug Administration.
  13. Brezinger-Dayan M, et al. CAR-T Stability 4h at 5 ± 3°C. PMC. 2022.
  14. Petrella A, et al. Fresh CAR-T Quality Attributes Stable 24h at 4°C. ScienceDirect. 2025.
  15. Luanpitpong S, et al. CAR-T Products Stable at 4°C for ≥24h in Study. PMC. 2024.
  16. FDA Guidance on Cord Blood Products: Validated Processes and Storage Quality Control. ebmt.org.
  17. Tran K, et al. HPC/Leukapheresis Stability: 4°C vs RT. ScienceDirect. 2024.
  18. Jansen J, et al. PBSC/CD34+ Stability During Cold Storage. Biomed Pharma Journal. 2009.
  19. Antonenas V, et al. PBSC Storage Temperature Impact. BioInsights Publishing. 2006.
  20. Nofianti L, et al. MSC Viability Differs by Storage Solution. Google Patents. 2018.
  21. Yuan Y, et al. Pluripotent Stem Cells: LN₂ Favored; –80°C Instability/Ice Recrystallization Risk. Nature (Scientific Reports). 2016.
  22. Dobruskin L. Freezing/Thawing Review: Storage Strategy Considerations. MDPI. 2024.

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