Advertisement

Cell and Tissue Banking

, Volume 16, Issue 2, pp 181–193 | Cite as

Defined serum- and xeno-free cryopreservation of mesenchymal stem cells

  • Shahla Hamza Al-SaqiEmail author
  • Mohammed Saliem
  • Hernan Concha Quezada
  • Åsa Ekblad
  • Aino Fianu Jonasson
  • Outi Hovatta
  • Cecilia Götherström
Original Paper

Abstract

Mesenchymal stem cells (MSCs) have vast potential in cell therapy, and are experimentally used in the clinic. Therefore, it is critical to find a serum- and xeno-free cryopreservation method. The aim of this study was to compare two serum- and xeno-free cryoprotectants for MSCs. Adipose tissue MSCs (Ad-MSCs) and bone marrow MSCs (BM-MSCs) were cryopreserved in two cryoprotectants: the defined serum- and xeno-free STEM-CELLBANKER™ (CB) and 10 % dimethyl sulfoxide (DMSO) in a xeno-free serum replacement cell culture medium and compared to non-cryopreserved MSCs. MSCs cryopreserved in CB or DMSO had similar morphology and surface marker expression compared to their respective non-cryopreserved MSC. Ad-MSCs and BM-MSC in both cryoprotectant media exhibited reduced mean fluorescence intensity (MFI) for CD105, BM-MSCs for CD73 and Ad-MSC increased MFI for HLA class I compared to non-cryopreserved MSCs. Population doubling time of CB cryopreserved and non-cryopreserved Ad-MSCs was similar (38.1 ± 13.6 and 36.8 ± 12.1 h), but somewhat higher when cryopreserved in DMSO (42.2 ± 10.8 h). BM-MSCs had higher population doubling time (CB 47.7 ± 11.4 and DMSO 62.3 ± 32.9 h respectively, p < 0.05) compared to Ad-MSCs. The viability of Ad-MSCs was significantly higher after cryopreservation in CB compared to DMSO (90.4 ± 4.5 % vs. 79.9 ± 3.8 % respectively). Ad-MSCs and BM-MSCs retained their mesodermal differentiation potential when cryopreserved in both cryoprotectants. The characteristics of Ad-MSCs post-thawing are better preserved by CB than by DMSO in serum- and xeno-free medium. Furthermore, Ad-MSCs and BM-MSCs are differently affected by the cryoprotectants, which may have implications for cell therapy.

Keywords

Mesenchymal stem cells Mesenchymal stromal cells Adipose Bone marrow Cryopreservation Serum- and xeno-free cryoprotectant STEM-CELLBANKER™ DMSO 

Abbreviations

MSCs

Mesenchymal stem cells

Ad-MSCs

Adipose tissue-derived MSCs

CB

STEM-CELLBANKER™

DMSO

Dimethyl sulfoxide

BM-MSCs

Bone marrow-derived MSCs

HBSS

Hanks balanced salt solution

PBS

Phosphate-buffered saline

LDA

Live/dead assay

PI

Propidium iodide

PDT

Population doubling time

DMEM-HG

Dulbecco’s modified eagles medium-high glucose

FBS

Fetal bovine serum

IBMX

Isobutyl-1-methylxanthine

Notes

Acknowledgments

This study has been supported by Karolinska Institutet foundation. We thank ZENOAQ for supplying the CB and CELLOTION media.

References

  1. Al-Saqi SH, Saliem M, Asikainen S, Quezada HC, Hovatta O, Le Blanc K et al (2014) Defined serum-free medium for in vitro expansion of adipose derived mesenchymal stem cells. Cytotherapy. doi: 10.1016/j.jcyt.2014.02.006 PubMedGoogle Scholar
  2. Basu J, Genheimer CW, Guthrie KI, Sangha N, Quinlan SF, Bruce AT et al (2011) Expansion of the human adipose-derived stromal vascular cell fraction yields a population of smooth muscle-like cells with markedly distinct phenotypic and functional properties relative to mesenchymal stem cells. Tissue Eng Part C Methods 17(8):843–860 Epub 2011/05/21CrossRefPubMedGoogle Scholar
  3. Bray LJ, Heazlewood CF, Atkinson K, Hutmacher DW, Harkin DG (2012) Evaluation of methods for cultivating limbal mesenchymal stromal cells. Cytotherapy 14(8):936–947 Epub 2012/05/17Google Scholar
  4. Campioni D, Lanza F, Moretti S, Ferrari L, Cuneo A (2008) Loss of Thy-1 (CD90) antigen expression on mesenchymal stromal cells from hematologic malignancies is induced by in vitro angiogenic stimuli and is associated with peculiar functional and phenotypic characteristics. Cytotherapy 10(1):69–82 Epub 2008/01/19CrossRefPubMedGoogle Scholar
  5. Casteilla L, Planat-Benard V, Laharrague P, Cousin B (2011) Adipose-derived stromal cells: their identity and uses in clinical trials, an update. World J Stem Cells 3(4):25–33 Epub 2011/05/25CrossRefPubMedCentralPubMedGoogle Scholar
  6. de Lima Prata K, de Santis GC, Orellana MD, Palma PV, Brassesco MS, Covas DT (2012) Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions. Cytotherapy 14(6):694–700 Epub 2012/04/24CrossRefPubMedGoogle Scholar
  7. Dicker A, Le Blanc K, Astrom G, van Harmelen V, Gotherstrom C, Blomqvist L et al (2005) Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 308(2):283–290 Epub 2005/06/01CrossRefPubMedGoogle Scholar
  8. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317 Epub 2006/08/23CrossRefPubMedGoogle Scholar
  9. Ginis I, Grinblat B, Shirvan MH (2012) Evaluation of bone marrow-derived mesenchymal stem cells after cryopreservation and hypothermic storage in clinically safe medium. Tissue Eng Part C Methods 18(6):453–463 Epub 2011/12/27CrossRefPubMedGoogle Scholar
  10. Halvorsen YD, Bond A, Sen A, Franklin DM, Lea-Currie YR, Sujkowski D et al (2001) Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis. Metabolism 50(4):407–413 Epub 2001/04/05CrossRefPubMedGoogle Scholar
  11. Holm F, Strom S, Inzunza J, Baker D, Stromberg AM, Rozell B et al (2010) An effective serum- and xeno-free chemically defined freezing procedure for human embryonic and induced pluripotent stem cells. Hum Reprod 25(5):1271–1279 Epub 2010/03/09CrossRefPubMedCentralPubMedGoogle Scholar
  12. Ikegame Y, Yamashita K, Hayashi S, Mizuno H, Tawada M, You F et al (2011) Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy. Cytotherapy 13(6):675–685 Epub 2011/01/15CrossRefPubMedGoogle Scholar
  13. Janz Fde L, Debes Ade A, Cavaglieri Rde C, Duarte SA, Romao CM, Moron AF, et al (2012) Evaluation of distinct freezing methods and cryoprotectants for human amniotic fluid stem cells cryopreservation. J Biomed Biotechnol 2012:649353. Epub 2012/06/06Google Scholar
  14. Kim WS, Park BS, Sung JH (2009) Protective role of adipose-derived stem cells and their soluble factors in photoaging. Arch Dermatol Res 301(5):329–336 Epub 2009/04/28CrossRefPubMedGoogle Scholar
  15. Le Blanc K (2006) Mesenchymal stromal cells: tissue repair and immune modulation. Cytotherapy 8(6):559–561 Epub 2006/12/07CrossRefPubMedGoogle Scholar
  16. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003) Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57(1):11–20 Epub 2003/01/25CrossRefPubMedGoogle Scholar
  17. Lin YT, Chern Y, Shen CK, Wen HL, Chang YC, Li H et al (2011) Human mesenchymal stem cells prolong survival and ameliorate motor deficit through trophic support in Huntington’s disease mouse models. PLoS ONE 6(8):e22924 Epub 2011/08/19CrossRefPubMedCentralPubMedGoogle Scholar
  18. Lin RZ, Moreno-Luna R, Zhou B, Pu WT, Melero-Martin JM (2012) Equal modulation of endothelial cell function by four distinct tissue-specific mesenchymal stem cells. Angiogenesis 15(3):443–455 Epub 2012/04/25CrossRefPubMedCentralPubMedGoogle Scholar
  19. Miwa H, Hashimoto Y, Tensho K, Wakitani S, Takagi M (2012) Xeno-free proliferation of human bone marrow mesenchymal stem cells. Cytotechnology 64(3):301–308. doi: 10.1007/s10616-011-9400-7
  20. Motta JPR, Paraguassú-Braga FH, Bouzas LF, Porto LC (2014) Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood. Cryobiology 68(3):343–348. doi: 10.1016/j.cryobiol.2014.04.007
  21. Naaldjik Y, Staude M, Federova V, Stolzing A (2012) Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide. BMC Biotechnol 12(1):49 Epub 2012/08/15CrossRefGoogle Scholar
  22. Pal R, Hanwate M, Jan M, Totey S (2009) Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow-derived mesenchymal stem cells. J Tissue Eng Regen Med 3(3):163–174 Epub 2009/02/21CrossRefPubMedGoogle Scholar
  23. Pegg DE (2007) Principles of cryopreservation. Methods Mol Biol 368:39–57 Epub 2007/12/18CrossRefPubMedGoogle Scholar
  24. Qi W, Ding D, Salvi RJ (2008) Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures. Hear Res 236(1–2):52–60 Epub 2008/01/22CrossRefPubMedCentralPubMedGoogle Scholar
  25. Saliem M, Holm F, Bergström Tengzelius R, Jorns C, Nilsson L-M, Ericzon B-G et al (2012) Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution. World J Hepatol 4(5):176CrossRefPubMedCentralPubMedGoogle Scholar
  26. Santos NC, Figueira-Coelho J, Saldanha C, Martins-Silva J (2002) Biochemical, biophysical and haemorheological effects of dimethylsulphoxide on human erythrocyte calcium loading. Cell Calcium 31(4):183–188 Epub 2002/05/25CrossRefPubMedGoogle Scholar
  27. Stiff PJ, Koester AR, Weidner MK, Dvorak K, Fisher RI (1987) Autologous bone marrow transplantation using unfractionated cells cryopreserved in dimethylsulfoxide and hydroxyethyl starch without controlled-rate freezing. Blood 70(4):974–978 Epub 1987/10/01PubMedGoogle Scholar
  28. Strem BM, Hicok KC, Zhu M, Wulur I, Alfonso Z, Schreiber RE et al (2005) Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med 54(3):132–141 Epub 2005/10/21CrossRefPubMedGoogle Scholar
  29. Witkowska-Zimny M, Walenko K (2011) Stem cells from adipose tissue. Cell Mol Biol Lett 16(2):236–257 Epub 2011/03/12CrossRefPubMedGoogle Scholar
  30. Zeisberger SM, Schulz JC, Mairhofer M, Ponsaerts P, Wouters G, Doerr D et al (2011) Biological and physicochemical characterization of a serum- and xeno-free chemically defined cryopreservation procedure for adult human progenitor cells. Cell Transpl 20(8):1241–1257 Epub 2010/12/24CrossRefGoogle Scholar
  31. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ et al (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7(2):211–228 Epub 2001/04/17CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Shahla Hamza Al-Saqi
    • 1
    Email author
  • Mohammed Saliem
    • 1
  • Hernan Concha Quezada
    • 2
  • Åsa Ekblad
    • 1
    • 3
  • Aino Fianu Jonasson
    • 1
  • Outi Hovatta
    • 1
  • Cecilia Götherström
    • 1
    • 3
  1. 1.Division of Obstetrics and Gynecology, K57, Department of Clinical Science, Intervention and TechnologyKarolinska Institutet, Karolinska University HospitalStockholmSweden
  2. 2.Center for Infectious Medicine, Department of MedicineKarolinska Institutet, Karolinska University HospitalHuddingeSweden
  3. 3.Center for Hematology and Regenerative MedicineKarolinska InstitutetStockholmSweden

Personalised recommendations