Skip to main content

The Role of Preservation in the Variability of Regenerative Medicine Products


Regenerative medicine (RM) has the potential to restore or establish normal function of cells, tissues, and organs that have been lost due to age, disease, or injury. It is common for the site of raw material collection, site of manufacture, and site of clinical use to be different for RM products, and at the same time, cells must remain viable and functional during transportation among different sites. Freezing products down to cryogenic temperatures along with cold chain transportation has become an effective method of preserving RM products. The quality of RM products along this supply chain represents the cumulative effects of all of the processing steps and all of the reagents used in the process. A variety of sources of variability in the preservation of RM products can result in both cell losses and greater variability in the quality of RM products. The purpose of this article is to review the sources of variability in the preservation process as well as the methods by which variability can be controlled or avoided.

Lay Summary

RM products involving the use of allogeneic or autologous cells and tissues are typically cryopreserved before shipping. Each step of the preservation process is a potential source of variability and can probably result in variability in the quality of RM products. This review provides an overview of the sources of variability in the processing of preservation and simple practices that can be used to control or reduce the variability. Future work will focus on incorporation of clinical outcomes with processing that has been performed to identify the root cause of quality inconsistency of RM products, as well as exploring the possibilities of simplifying and automating processing steps of preservation in order to reduce the overall variability that might be present.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3


  1. Bayon Y, Vertès AA, Ronfard V, Culme-Seymour E, Mason C, Stroemer P, et al. Turning regenerative medicine breakthrough ideas and innovations into commercial products. Tissue Eng Part B Rev. 2015;21:560–71.

    Article  Google Scholar 

  2. Blair NF, Frith TJR, Barbaric I. Regenerative medicine: advances from developmental to degenerative diseases. 2017 [cited 2019 Apr 28]. p. 225–239. Available from:

  3. Corbett MS, Webster A, Hawkins R, Woolacott N. Innovative regenerative medicines in the EU: a better future in evidence? BMC Med. 2017;15:49.

    Article  Google Scholar 

  4. Gee AP. Regulation of regenerative medicine products. 2018 [cited 2019 Apr 28]. p. 189–198. Available from:

  5. Muraca M, Piccoli M, Franzin C, Tolomeo A, Jurga M, Pozzobon M, et al. Diverging concepts and novel perspectives in regenerative medicine. Int J Mol Sci. 2017;18.

  6. Osakada F. Development of cellular and tissue-based products for retinal regenerative medicine. Yakugaku Zasshi. 2017;137:23–9.

    CAS  Article  Google Scholar 

  7. Richards MM, Maxwell JS, Weng L, Angelos MG, Golzarian J. Intra-articular treatment of knee osteoarthritis: from anti-inflammatories to products of regenerative medicine. Phys Sportsmed. 2016;44:101–8.

    Article  Google Scholar 

  8. Terzic A, Pfenning MA, Gores GJ, Harper CM. Regenerative medicine build-out. Stem Cells Transl Med. 2015;4:1373–9.

    Article  Google Scholar 

  9. Dzobo K, Thomford NE, Senthebane DA, Shipanga H, Rowe A, Dandara C, et al. Advances in regenerative medicine and tissue engineering: innovation and transformation of medicine. Stem Cells Int. 2018;2018:1–24.

    Article  CAS  Google Scholar 

  10. Hoffman T, Khademhosseini A, Langer RS. Chasing the paradigm: clinical translation of 25 years of tissue engineering. Tissue Eng Part A. 2019.

  11. Hubel A. Impact of freeze–thaw processes on the quality of cells. Cell Gene Ther Insights. 2017;3:807–13.

    Article  Google Scholar 

  12. Hubel A, Carlquist D, Clay M, McCullough J. Liquid storage, shipment, and cryopreservation of cord blood. Transfusion. 2004;44:518–25.

    Article  Google Scholar 

  13. François MM, Copland IB, Yuan S, Romieu-Mourez RR, Waller EK, Galipeau J. Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing. Cytotherapy. 2012;14:147–52.

    Article  CAS  Google Scholar 

  14. Hubel A. Preservation of cells: a practical manual. Hoboken, NJ: John Wiley & Sons; 2018.

    Book  Google Scholar 

  15. Freedman L. Reproducibility: life sciences lag in stringent standards. Nature. 2013;504:376.

    CAS  Article  Google Scholar 

  16. Betsou F, Lehmann S, Ashton G, Barnes M, Benson EE, Coppola D, et al. Standard preanalytical coding for biospecimens; defining the sample PREanalytical code. Cancer Epidemiol Biomark Prev. 2010;19:1004–11.

    CAS  Article  Google Scholar 

  17. Areman E. Cellular therapy: principles, methods, and regulations; 2009. p. 236–50.

    Google Scholar 

  18. Pollock K, Sumstad D, Kadidlo D, McKenna DH, Hubel A. Clinical mesenchymal stromal cell products undergo functional changes in response to freezing. Cytotherapy. 2015;17:38–45.

    Article  Google Scholar 

  19. Pi CH, Yu G, Petersen A, Hubel A. Characterizing the “sweet spot” for the preservation of a T-cell line using osmolytes. Sci Rep. 2018;8:16223.

    Article  CAS  Google Scholar 

  20. Solocinski J, Osgood Q, Wang M, Connolly A, Menze, A. M, Chakraborty N. Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation, cellular viability, and metabolism. Cryobiology. 2017 [cited 2018 May 9];75:134–143. Available from:

  21. Castro VIB, Craveiro R, Silva JM, Reis RL, Paiva A, Ana AR. Natural deep eutectic systems as alternative nontoxic cryoprotective agents. Cryobiology. 2018;83:15–26.

    CAS  Article  Google Scholar 

  22. Yu G, Li R, Hubel A. Interfacial interactions of sucrose during cryopreservation detected by Raman spectroscopy. Langmuir. 2018 [cited 2019 Apr 28];acs.langmuir.8b01616. Available from:

  23. Crowe JH, Carpenter JF, Crowe LM. The role of vitrification in anhydrobiosis. Annu Rev Physiol. 1998;60:73–103 Available from:

    CAS  Article  Google Scholar 

  24. Sputtek A, Körber C. Cryopreservation of red blood cells, platelets, lymphocytes, and stem cells. Boca Raton: CRC Press; 1991.

    Google Scholar 

  25. Fahy GM. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology. 1986;23:1–13.

    CAS  Article  Google Scholar 

  26. Levin RL, Miller TW. An optimum method for the introduction or removal of permeable cryoprotectants: isolated cells. Cryobiology. 1981.

  27. FRASER JK, CAIRO MS, WAGNER EL, McCURDY PR, BAXTER-LOWE LA, CARTER SL, et al. Cord Blood Transplantation Study (COBLT): cord blood bank standard operating procedures. J Hematother. 1998 [cited 2019 Apr 27];7:521–61. Available from:

  28. Reich-Slotky R, Bachegowda LS, Ancharski M, Mendeleyeva L, Rubinstein P, Rennert H, et al. How we handled the dextran shortage: an alternative washing or dilution solution for cord blood infusions. Transfusion. 2015;55:1147–53.

    CAS  Article  Google Scholar 

  29. Hanna J, Hubel A. Preservation of stem cells. Organogenesis. 2009;5:134–7.

    Article  Google Scholar 

  30. Pi C-H, Yu G, Dosa PI, Hubel A. Characterizing modes of action and interaction for multicomponent osmolyte solutions on Jurkat cells. Biotechnol Bioeng 2018 [cited 2019 Jan 4]; Available from:

  31. Pollock K, Yu G, Moller-Trane R, Koran M, Dosa PI, McKenna DH, et al. Combinations of osmolytes, including monosaccharides, disaccharides, and sugar alcohols act in concert during cryopreservation to improve mesenchymal stromal cell survival. Tissue Eng Part C Methods. 2016;22:999–1008.

    CAS  Article  Google Scholar 

  32. Mazur P, Leibo SP, Farrant J, Chu EHY, Hanna MG, Smith LH. Interactions of cooling rate, warming rate and protective additive on the survival of frozen mammalian cells. 2008 [cited 2019 Jan 2]. p. 69–88. Available from:

  33. Mazur P. Principles of cryobiology. Life frozen state: CRC Press; 2004. p. 3–66.

  34. Buckler RL, Kunkel EJ, Thompson ML, Ehrhardt RO. Technological developments for small-scale downstream processing of cell therapies. Cytotherapy. 2016;18:301–6.

    CAS  Article  Google Scholar 

  35. Lopez E, Cipri K, Cryobiology VN-C frontiers in, 2012 undefined. Technologies for cryopreservation: overview and innovation. [cited 2018 Dec 29]; Available from:

  36. Katayama Y, Yano T, Bessho A, Deguchi S, Sunami K, Mahmut N, et al. The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells. Bone Marrow Transplant. 1997;19:283–7.

    CAS  Article  Google Scholar 

  37. Li R, Yu G, Azarin SM, Hubel A. Freezing responses in DMSO-based cryopreservation of human iPS cells: aggregates versus single cells. Tissue Eng Part C Methods. 2018 [cited 2018 Apr 8];24. Available from:

  38. Harris CL, Toner M, Hubel A, Cravalho EG, Yarmush ML, Tompkins RG. Cryopreservation of isolated hepatocytes: intracellular ice formation under various chemical and physical conditions. Cryobiology. 1991;28:436–44 Available from:

    CAS  Article  Google Scholar 

  39. Diller KR. Intracellular freezing: effect of extracellular supercooling. Cryobiology. 1975;12:480–5.

    CAS  Article  Google Scholar 

  40. Karantalis V, Schulman IH, Balkan W, Hare JM. Allogeneic cell therapy: a new paradigm in therapeutics. Circ Res. 2015;116:12–5.

    CAS  Article  Google Scholar 

  41. Pigeau GM, Csaszar E, Dulgar-Tulloch A. Commercial scale manufacturing of allogeneic cell therapy. Front Med. 2018;5 Available from:

  42. Tedder RS, Zuckerman MA, Brink NS, Goldstone AH, Fielding A, Blair S, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet. 1995;346:137–40.

    CAS  Article  Google Scholar 

  43. Cosentino LM, Corwin W, Baust JM, Diaz-Mayoral N, Cooley H, Shao W, et al. Preliminary report: evaluation of storage conditions and cryococktails during peripheral blood mononuclear cell cryopreservation. Cell Preserv Technol. 2007;5:189–204 Available from:

    Article  Google Scholar 

  44. Hubel A, Spindler R, Curtsinger JM, Lindgren B, Wiederoder S, McKenna DH. Postthaw characterization of umbilical cord blood: markers of storage lesion. Transfusion. 2015;55:1033–9.

    CAS  Article  Google Scholar 

  45. Campbell LD, Astrin JJ, DeSouza Y, Giri J, Patel AA, Rawley-Payne M, et al. The 2018 revision of the ISBER best practices : summary of changes and the editorial team’s development process. Biopreserv Biobank. 2018;16:3–6.

    Article  Google Scholar 

  46. USP. National formulary standards for biologics cryopreservation of cells. Washington, D.C.: US Pharmacop; 2014. p. 1–31.

    Google Scholar 

  47. Karlsson JO. A theoretical model of intracellular devitrification. Cryobiology. 2001;42:154–69 Available from:

    CAS  Article  Google Scholar 

  48. Antonenas V, Bradstock KF, Shaw PJ. Effect of washing procedures on unrelated cord blood units for transplantation in children and adults. Cytotherapy. 2002;4:16.

    Google Scholar 

  49. Zhou X, Liu Z, Shu Z, Ding W, Du P, Chung J, et al. A dilution-filtration system for removing cryoprotective agents. J Biomech Eng. 2011;133:021007 Available from:

    Article  Google Scholar 

  50. Tostoes R, Dodgson JR, Weil B, Gerontas S, Mason C, Veraitch F. A novel filtration system for point of care washing of cellular therapy products. J Tissue Eng Regen Med. 2017;11:3157–67.

    CAS  Article  Google Scholar 

  51. Hanna J, Hubel A, Lemke E. Diffusion-based extraction of DMSO from a cell suspension in a three stream, vertical microchannel. Biotechnol Bioeng. 2012;109:2316–24.

    CAS  Article  Google Scholar 

  52. Pegg DE. Viability assays for preserved cells, tissues, and organs. Cryobiology. 1989;26:212–31.

    CAS  Article  Google Scholar 

  53. Ragoonanan V, Hubel A, Aksan A. Response of the cell membrane–cytoskeleton complex to osmotic and freeze/thaw stresses. Cryobiology. Academic Press; 2010 [cited 2019 Apr 27];61:335–44. Available from:

  54. Hubel A. Cellular preservation: gene therapy cellular metabolic engineering. Adv Biopreservation. 2006.

  55. Diepart C, Verrax J, Calderon PB, Feron O, Jordan BF, Gallez B. Comparison of methods for measuring oxygen consumption in tumor cells in vitro. Anal Biochem. 2010;396:250–6.

    CAS  Article  Google Scholar 

  56. Liu Y, Peterson D, Hideo K, David S. Mechanism of cellular 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem. 1997;69:581–93 Available from:

    CAS  Article  Google Scholar 

  57. Cui D, Li H, Wan M, Peng Y, Xu X, Zhou X, et al. The origin and identification of mesenchymal stem cells in teeth: from odontogenic to non-odontogenic. Curr Stem Cell Res Ther. 2017;13.

  58. Fazeli Z, Abedindo A, Omrani MD, Ghaderian SMH. Mesenchymal stem cells (MSCs) therapy for recovery of fertility: a systematic review. Stem Cell Rev Reports. 2018 [cited 2019 Apr 28];14:1–12. Available from:

  59. Gardner OFW, Alini M, Stoddart MJ. Mesenchymal stem cells derived from human bone marrow. 2015 [cited 2019 Apr 28]. p. 41–52. Available from:

  60. Dzobo K, Turnley T, Wishart A, 2016 Fibroblast-derived extracellular matrix induces chondrogenic differentiation in human adipose-derived mesenchymal stromal/stem cells in vitro. [cited 2019 Apr 28]; Available from:

  61. Lou G, Chen Z, Zheng M, Liu Y. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Exp Mol Med. 2017.

  62. Mao F, Tu Q, Wang L, Chu F, Li X, Li H, et al. Mesenchymal stem cells and their therapeutic applications in inflammatory bowel disease. [cited 2019 Apr 28]; Available from:

  63. Mohr A, Zwacka R. The future of mesenchymal stem cell-based therapeutic approaches for cancer—from cells to ghosts. Cancer Lett. 2018;414:239–49.

    CAS  Article  Google Scholar 

  64. Moreira A, 2017. Therapeutic potential of mesenchymal stem cells for diabetes. [cited 2019 Apr 28]; Available from:

  65. Park WS, Ahn SY, Sung SI, Ahn J-Y, Chang YS. Mesenchymal stem cells: the magic cure for intraventricular hemorrhage? Cell Transplant. 2017;26:439–48.

    Article  Google Scholar 

  66. Dzobo K, Vogelsang M, Parker MI. Wnt/β-catenin and MEK-ERK signaling are required for fibroblast-derived extracellular matrix-mediated endoderm differentiation of embryonic stem cells. Stem Cell Rev Reports. 2015 [cited 2019 Apr 28];11:761–773. Available from:

  67. Fahy GM, Wowk B. Principles of cryopreservation by vitrification. Methods Mol Biol. 2015.

Download references


This work was funded in part by R01EB023880.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Allison Hubel.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yu, G., Hubel, A. The Role of Preservation in the Variability of Regenerative Medicine Products. Regen. Eng. Transl. Med. 5, 323–331 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Cryopreservation
  • Regenerative medicine