A Freezing Protocol for Hematopoietic Stem Cells

  • Petra PavelEmail author
  • Sascha Laier
Part of the Methods in Molecular Biology book series (MIMB, volume 2017)


Especially in the field of autologous transplantation, it has been found necessary to develop methods that ensure long-term storage with maintenance of functionality of the cells to bridge the therapy-related temporal separation of collection and application.

Based on the experiences of more than 40 years, some practical considerations, especially regarding the cell concentration, final volume, and possibly other exogenous substances, should be considered when establishing a protocol for the routine cryopreservation of peripheral blood stem cells. In the following chapter, we describe a freezing protocol for cryopreservation of peripheral blood stem cells which was used and optimized over the past 8 years and was applied to the cryopreservation of more than 2000 peripheral stem cell transplants.

Key words

Cryopreservation Cryoprotectants Autologous peripheral blood stem cells Autologous stem cell transplantation 


  1. 1.
    Gonzales F, Luyet B (1950) Resumption of heart-beat in chick embryo frozen in liquid nitrogen. Biodynamica 7:1–5PubMedGoogle Scholar
  2. 2.
    Schöpf-Ebner E, Gross WO, Bucher OM (1968) Pulsatile activity of isolated heart muscle cells after freezing storage. Cryobiology 4:200–203CrossRefGoogle Scholar
  3. 3.
    Mazur P (1965) Causes of injury in frozen and thawed cells. Fed Proc 24:S175–S182PubMedGoogle Scholar
  4. 4.
    Mazur P (1970) Cryobiology: the freezing of biological systems. Science 168:939–949CrossRefGoogle Scholar
  5. 5.
    Morris GJ, Acton E (2013) Controlled ice nucleation in cryopreservation—a review. Cryobiology 66:85–92CrossRefGoogle Scholar
  6. 6.
    Karow AM Jr (1969) Biological effects of cryoprotectants as related to cardiac cryopreservation. Cryobiology 5:429–443CrossRefGoogle Scholar
  7. 7.
    Karow AM Jr (1969) Cryoprotectants—a new class of drugs. J Pharm Pharmacol 21:209–223CrossRefGoogle Scholar
  8. 8.
    Karow AM Jr (1974) Cryopreservation: pharmacological considerations. In: Karow AMS, Karow GJM, Humphries AI (eds) Organ preservation for transplantation. Little Brown & Co, Boston, pp 86–107Google Scholar
  9. 9.
    Ashwood-Smith MJ (1987) Mechanisms of cryoprotectant action. Symposia Soc Exp Biol 41:395–406Google Scholar
  10. 10.
    Elliott GD, Wang S, Fuller BJ (2017) A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology 76:74–91CrossRefGoogle Scholar
  11. 11.
    Hubalek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46:205–229CrossRefGoogle Scholar
  12. 12.
    Berz D, McCormack EM, Winer ES, Colvin GA, Quesenberry PJ (2007) Cryopreservation of hematopoietic stem cells. Am J Hematol 82:1–8CrossRefGoogle Scholar
  13. 13.
    Choi CW, Kim BS, Shin SW, Kim YH, Kim JS (2007) Long-term engraftment stability of peripheral blood cells cryopreserved using the dump-freezing method in a −80°C mechanical freezer with 10% dimethyl sulfoxide. Int J Hematol 3:245–250Google Scholar
  14. 14.
    Arakawa T, Carpenter JF, Kita YA, Crowe JH (1990) The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 27:401–415CrossRefGoogle Scholar
  15. 15.
    Günzel P, Eichler H (2008) Zusammenfassung und Bewertung der toxiko-pharmakologischen Daten und Informationen zu Dimethylsulfoxid (DMSO) im Hinblick auf seine Verwendung in Stammzellzubereitungen als gutachterliche Ergänzung der Gemeinsamen Stellungnahme der Fachgesellschaften DGTI, DGHO und GPOH zu Genehmigungsverfahren von Stammzellzubereitungen (
  16. 16.
    Kim KM, Huh JY, Kim JJ, Kang MS (2017) Quality comparison of umbilical cord blood cryopreserved with conventional versus automated systems. Cryobiology 78:65–69CrossRefGoogle Scholar
  17. 17.
    Li L, Chen Z, Zhang M, Panhwar F, Gao C, Zhao G, Jin B, Ye B (2017) Cell membrane permeability coefficients determined by single-step osmotic shift are not applicable for optimization of multi-step addition of cryoprotective agents: as revealed by HepG2 cells. Cryobiology 79:82–86CrossRefGoogle Scholar
  18. 18.
    Freimark D, Sehl C, Weber C, Hudel K, Czermak P, Hofmann N, Spindler R, Glasmacher B (2011) Systematic parameter optimization of a ME2SO- and serum-free cryopreservation protocol for human mesenchymal stem cells. Cryobiology 63:67–75CrossRefGoogle Scholar
  19. 19.
    Son JH, Heo YJ, Park MY, Kim HH, Lee KS (2010) Optimization of cryopreservation condition for hematopoietic stem cells from umbilical cord blood. Cryobiology 60:287–292CrossRefGoogle Scholar
  20. 20.
    Hopkins JB, Badeau R, Warkentin M, Thorne RE (2012) Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions. Cryobiology 65:169–178CrossRefGoogle Scholar
  21. 21.
    Shleback AA, Marley SB, Roberts IA, Davidson RJ, Goldman JM, Gordon MY (1999) Optimal timing for processing and cryopreservation of umbilical cord hematopoietic stem cells for clinical transplantation. Bone Marrow Transplant 23:131–136CrossRefGoogle Scholar
  22. 22.
    Johnson LN, Winter KM, Reid S, Hartkopf-Theis T, Marks DC (2011) Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution. Cryobiology 62:100–106CrossRefGoogle Scholar
  23. 23.
    Solves P, Mirabet V, Planelles D, Carbonell-Uberos F, Roig R (2008) Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation. Cryobiology 56:152–158CrossRefGoogle Scholar
  24. 24.
    Best A, Hidalgo G, Mitchell K, Yanelli JR (2007) Issues concerning the large scale cryopreservation of peripheral blood mononuclear cells (PBMC) for immunotherapy trials. Cryobiology 54:294–297CrossRefGoogle Scholar
  25. 25.
    Yang H, Zhao H, Acker JP, Liu JZ, Akabutu J, McGann LE (2005) Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood. Cryobiology 51:165–175CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Stem Cell LaboratoryInstitute of Clinical Transfusion Medicine and Cell Therapy Heidelberg GmbHHeidelbergGermany

Personalised recommendations