Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study

Theodros Mamo¹, Cheryl A Cox², Connor Demorest³, Magali J Fontaine⁴, Allison Hubel⁵, Linda Kelley², Aisha Khan⁶, Denese C Marks⁷, Shibani Pati⁸, Jo-Anna Reems⁹, Gabriele Spohn¹⁰, Richard Schäfer¹¹, Rongye Shi¹², Lipei Shao¹², David Stroncek¹², David H McKenna¹³; Biomedical Excellence for Safer Transfusion (BEST) Collaborative

Affiliations

¹ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA. Electronic address: mamo0016@umn.edu.
² Moffitt Cancer Center, Tampa, Florida, USA.
³ Masonic Cancer Center Biostatistics Core, University of Minnesota, Minneapolis, Minnesota, USA.
⁴ University of Maryland School of Medicine, Baltimore, Maryland, USA.
⁵ Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, USA; Evia Bio, Minneapolis, Minnesota, USA.
⁶ University of Miami, Coral Gables, Florida, USA.
⁷ Research and Development, Australian Red Cross Lifeblood, Sydney, Australia.
⁸ University of California San Francisco, San Francisco, California, USA.
⁹ University of Utah, Salt Lake City, Utah, USA.
¹⁰ German Red Cross Blood Donor Service and Goethe University Hospital, Frankfurt am Main, Germany.
¹¹ German Red Cross Blood Donor Service and Goethe University Hospital, Frankfurt am Main, Germany; Medical Center, Center for Chronic Immunodeficiency, University of Freiburg, Freiburg, Germany.
¹² Center for Cellular Engineering, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
¹³ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA; Molecular and Cellular Therapeutics, University of Minnesota, Saint Paul, Minnesota, USA.

PMID: 39066775
PMCID: PMC11841823 (available on 2025-12-01)
DOI: 10.1016/j.jcyt.2024.07.001

Multicenter Study

Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study

Theodros Mamo et al. Cytotherapy. 2024 Dec.

. 2024 Dec;26(12):1522-1531.

doi: 10.1016/j.jcyt.2024.07.001. Epub 2024 Jul 6.

Authors

Affiliations

¹ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA. Electronic address: mamo0016@umn.edu.
² Moffitt Cancer Center, Tampa, Florida, USA.
³ Masonic Cancer Center Biostatistics Core, University of Minnesota, Minneapolis, Minnesota, USA.
⁴ University of Maryland School of Medicine, Baltimore, Maryland, USA.
⁵ Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, USA; Evia Bio, Minneapolis, Minnesota, USA.
⁶ University of Miami, Coral Gables, Florida, USA.
⁷ Research and Development, Australian Red Cross Lifeblood, Sydney, Australia.
⁸ University of California San Francisco, San Francisco, California, USA.
⁹ University of Utah, Salt Lake City, Utah, USA.
¹⁰ German Red Cross Blood Donor Service and Goethe University Hospital, Frankfurt am Main, Germany.
¹¹ German Red Cross Blood Donor Service and Goethe University Hospital, Frankfurt am Main, Germany; Medical Center, Center for Chronic Immunodeficiency, University of Freiburg, Freiburg, Germany.
¹² Center for Cellular Engineering, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
¹³ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA; Molecular and Cellular Therapeutics, University of Minnesota, Saint Paul, Minnesota, USA.

PMID: 39066775
PMCID: PMC11841823 (available on 2025-12-01)
DOI: 10.1016/j.jcyt.2024.07.001

Abstract

Background and aim: An essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs.

Methods: A DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5-10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis.

Results: MSCs had an average viability of 94.3% (95% CI: 87.2-100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03-9.0%; P: 0.049) and 11.4% (95% CI: 6.9-15.8%; P< 0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7-100.0%), and it was lower by 5.6% (95% CI: 1.3-9.8%, P < 0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90, and CD105 with no significant difference in global gene expression profiles.

Conclusion: MSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution.

Keywords: DMSO; cryopreservation; cryoprotectant; mesenchymal stem/stromal cells (MSCs).

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Conflict of interest statement

Declaration of competing interest AH is the Founder and Chief Scientific Officer of Evia Bio, a company that was established after the current study was completed and is commercializing the proprietary SGI solution used in this study.

References

1. Introna M, Lucchini G, Dander E, Galimberti S, Rovelli A, Balduzzi A, Longoni D, Pavan F, Masciocchi F, Algarotti A, Mico C, Grassi A, Deola S, Cavattoni I, Gaipa G, Belotti D, Perseghin P, Parma M, Pogliani E, Golay J, Pedrini O, Capelli C, Cortelazzo S, D’Amico G, Biondi A, Rambaldi A, Biagi E, Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients, Biol Blood Marrow Transplant 20(3) (2014) 375–81. - PubMed
1. Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringden O, Developmental B. Committee of the European Group for, T. Marrow, Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study, Lancet 371(9624) (2008) 1579–86. - PubMed
1. Yun CW, Lee SH, Enhancement of Functionality and Therapeutic Efficacy of Cell-Based Therapy Using Mesenchymal Stem Cells for Cardiovascular Disease, Int J Mol Sci 20(4) (2019). - PMC - PubMed
1. Lalu MM, Montroy J, Dowlatshahi D, Hutton B, Juneau P, Wesch N, Y.Z. S R. McGinn D. Corbett DJ Stewart AFD, From the Lab to Patients: a Systematic Review and Meta-Analysis of Mesenchymal Stem Cell Therapy for Stroke, Transl Stroke Res 11(3) (2020) 345–364. - PubMed
1. Matthay MA, Calfee CS, Zhuo H, Thompson BT, Wilson JG, Levitt JE, Rogers AJ, Gotts JE, Wiener-Kronish JP, Bajwa EK, Donahoe MP, McVerry BJ, Ortiz LA, Exline M, Christman JW, Abbott J, Delucchi KL, Caballero L, McMillan M, McKenna DH, Liu KD, Treatment with allogeneic mesenchymal stromal cells for moderate to severe acute respiratory distress syndrome (START study): a randomised phase 2a safety trial, Lancet Respir Med 7(2) (2019) 154–162. - PMC - PubMed

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Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study

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Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study

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