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Superior Cryopreservation of T Cells in Low-DMSO, Animal-Origin-Free Formulation Using Optibumin 25 and PentaHibe Base

Published on 21 August 2025

Application Note

Authors:  Andrew Hamann, PhD and Mark Stathos, PhD, InVitria, Inc., USA
In collaboration with PentaHibe, developed by Pharmacosmos A/S, Denmark

Overview

This application note presents a defined, animal-origin-free (AOF) cryopreservation strategy addressing the limitations of high-DMSO and serum-derived components. The formulation—10% PentaHibe Base, 2% DMSO, and 4% Optibumin 25—was tested in CAR-T–mimicking workflows using healthy donor T cells.

PentaHibe Base is a non-penetrating, carbohydrate-based cryoprotectant derived from pentaisomaltose, a patented subfraction of Dextran 1 with a well-established clinical safety profile. Optibumin 25 is a recombinant human serum albumin (HSA) produced under cGMP in a non-mammalian system.

The formulation was benchmarked against plasma-derived HSA and CryoStor CS10, CS5, and CS2. Despite using significantly less DMSO than CS10 and CS5—and the same concentration as CS2—it matched or exceeded post-thaw viability and proliferation across all donors and endpoints. These results support the use of this fully defined, animal-origin-free formulation in GMP-aligned cryopreservation workflows for adoptive cell therapies, including CAR-T.

Key Findings

  • Maintains high post-thaw viability with only 2% DMSO
  • Matches or exceeds CS10 and plasma-derived HSA
  • Outperforms CS2 across all donors and endpoints
  • Supports strong T cell proliferation at 72 hours
  • Defined, animal-origin-free composition suitable for GMP workflows

Featured Solutions

Optibumin 25 – Recombinant Human Albumin for Low-DMSO Cryopreservation

Optibumin 25 familyOptibumin 25 is a recombinant, animal-origin-free human serum albumin (rHSA) that replaces plasma-derived HSA in cryopreservation workflows. In this study, Optibumin 25 supported high post-thaw T cell viability and proliferation at only 2% DMSO, performing comparably to CryoStor CS10 and CS5 and significantly better than CS2. By eliminating variability and safety risks linked to blood-derived albumin, Optibumin 25 provides a defined, consistent, and regulatory-friendly solution for T cell and CAR-T manufacturing

Learn more about Optibumin 25

 

PentaHibe® Base – Carbohydrate-Based Cryoprotectant

PentaHibe BasePentaHibe Base is a carbohydrate-based cryoprotectant developed by Pharmacosmos A/S. Derived from pentaisomaltose, a patented Dextran 1 subfraction, PentaHibe acts extracellularly to protect cells during freeze–thaw cycles while minimizing intracellular toxicity. In this study, PentaHibe Base combined with Optibumin 25 enabled robust T cell viability and proliferation with only 2% DMSO, supporting safer and more scalable workflows for adoptive cell therapies

Learn more about PentaHibe Base

 

Frequently Asked Questions (FAQs)

Q: Why is low-DMSO important in T cell cryopreservation?
A: High concentrations of DMSO are cytotoxic and can cause infusion-related side effects. Using just 2% DMSO with Optibumin 25 and PentaHibe Base maintains high T cell viability while reducing toxicity.

Q: How does Optibumin 25 improve cryopreservation compared to plasma-derived HSA?
A: Optibumin 25 is recombinant human serum albumin produced without animal or human plasma. It eliminates donor variability and pathogen risk while supporting equal or better post-thaw recovery of T cells.

Q: What makes this cryopreservation method animal-origin-free?
A: The formulation replaces plasma-derived HSA with Optibumin 25 and pairs it with PentaHibe Base, a carbohydrate cryoprotectant. Both are produced under GMP conditions without animal inputs, supporting regulatory compliance.

Q: Is this method compatible with CAR-T cell therapy workflows?
A: Yes. The formulation was tested in CAR-T–mimicking workflows using healthy donor T cells and demonstrated strong post-thaw viability and proliferation at clinically relevant conditions.

Q: Does reducing DMSO affect T cell proliferation after thawing?
A: No. Even at 2% DMSO, T cells cryopreserved with Optibumin 25 and PentaHibe Base matched or exceeded proliferation seen with 10% DMSO CryoStor formulations.

References

  1. Burnham, R. E., et al. (2021). Human serum albumin and chromatin condensation rescue ex vivo expanded γδ T cells from the effects of cryopreservation. Cryobiology, 99, 78–87. https://doi.org/10.1016/j.cryobiol.2021.01.011
  2. Haastrup, E. K., et al. (2021). DMSO (Me2SO) concentrations of 1–2% in combination with pentaisomaltose are effective for cryopreservation of T cells. Transfusion and Apheresis Science, 60(4), 103138. https://doi.org/10.1016/j.transci.2021.103138
  3. InVitria. (2025). Low-DMSO cryopreservation of iPSCs with Optibumin® 25 recombinant albumin [Application Note]. https://invitria.com/resources/low-dmso-cryopreservation-of-ipscs-with-optibumin-25/
  4. InVitria. (2024). Enhanced cryopreservation of T cells using Optibumin® rHSA [Application Note]. https://invitria.com/resources/enhanced-cryopreservation-of-t-cells-using-optibumin-rhsa/
  5. InVitria. (2025). Low-DMSO cryopreservation of MSCs with Optibumin® 25 recombinant albumin [Application Note]. https://invitria.com/resources/low-dmso-cryopreservation-of-mscs-with-optibumin-25-recombinant-albumin/
  6. Li, R., et al. (2019). Preservation of cell-based immunotherapies for clinical trials. Cytotherapy, 21(9), 943–957. https://doi.org/10.1016/j.jcyt.2019.07.004
  7. Lu, H., Zhang, Y., & Liu, P. (2024). Identifying new safety risk of human serum albumin: A retrospective study of real-world data. Frontiers in Pharmacology, 15, 1319900. https://doi.org/10.3389/fphar.2024.1319900
  8. Ljungström, K. G. (2006). Invited commentary: Pretreatment with dextran 1 makes dextran 40 therapy safer. Journal of Vascular Surgery, 43(5), 1070–1072. https://doi.org/10.1016/j.jvs.2005.11.056
  9. MacLennan, S., & Barbara, J. A. J. (2006). Risks and side effects of therapy with plasma and plasma fractions. Best Practice & Research Clinical Haematology, 19(1), 169–189. https://doi.org/10.1016/j.beha.2005.01.033
  10. Madsen, B. K., et al. (2018). Adverse reactions of dimethyl sulfoxide in humans: A systematic review. F1000Research, 7. https://doi.org/10.12688/f1000research.16642.2
  11. U.S. Food and Drug Administration. (2024). Considerations for the use of human- and animal-derived materials in the manufacture of cell and gene therapy and tissue-engineered medical products. FDA-2024-D-1244. https://www.regulations.gov/docket/FDA-2024-D-1244
  12. Pharmacosmos A/S. (2024). DMSO-reduced cryopreservation using 10% PentaHibe® Base, 2% DMSO and 4% human albumin. https://pentahibe.com/dataAndDocuments
  13. Pi, C.-H., et al. (2020). Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants. Cytotherapy, 22(5), 291–300. https://doi.org/10.1016/j.jcyt.2020.01.013

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