FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Stem cell research & therapy2015; 6; 231; doi: 10.1186/s13287-015-0230-y

Cryopreservation of equine mesenchymal stem cells in 95% autologous serum and 5% DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20 or 95% and DMSO at 10 or 5.

Abstract: Equine superficial digital flexor tendon injury is a well-accepted model of human tendon injury and is routinely treated with local injections of autologous mesenchymal stem cells (MSCs). Identification of a clinically safe medium for short-term cryopreservation of MSCs prior to cell implantation would streamline laboratory and clinical procedures for autologous regenerative therapies. Veterinary experience with short-term (MSCs prepared after the injury has occurred) cryopreserved MSCs in naturally occurring injury in the horse will be of value to human practitioners. Methods: Equine bone marrow derived MSCs were cryopreserved in 6 different solutions consisting of 20% serum, 10% DMSO and 70% media or 95% serum and 5% DMSO. Serum was autologous serum, commercially available pooled equine serum or fetal bovine serum (FBS). Cell survival, morphology and growth kinetics were assessed by total cell number, measurement of growth kinetics, colony-forming-unit-assay and morphology of MSCs after monolayer culture post-thaw. Results: There were no significant differences in post-thaw viability, total cell number, morphology scores or growth kinetics among the 6 solutions. Post thaw viabilities from each group ranged from 80-90%. In all solutions, there were significantly fewer MSCs and the majority (99%) of MSCs remained in the original generation 24 hours post-thaw. Seventy two hours post-thaw, the majority of MSCs (50%) were proliferating in the fourth generation. Mean colony count in the CFU-F assay ranged from 72 to 115 colonies. Conclusions: Each of the serum sources could be used for short-term cryopreservation of equine bone marrow derived MSCs. Prior to clinical use, clinicians may prefer autologous serum and a lower concentration of DMSO.
Get Full Text
Publication Date: 2015-11-26 PubMed ID: 26611913PubMed Central: PMC4661990DOI: 10.1186/s13287-015-0230-yGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Comparative Study
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research study explores the effects of cryopreservation on equine mesenchymal stem cells (MSCs). The research concludes that freezing these cells in solutions containing 95% autologous serum (serum obtained from the same individual) and 5% DMSO (dimethyl sulfoxide) does not alter their growth or morphology compared to those cryopreserved with other types of serum or higher concentrations of DMSO.

Methodology

  • The MSCs in the study were taken from equine bone marrow, which has been commonly used in regenerative therapies to treat horse tendon injuries.
  • The researchers cryopreserved these MSCs in six different solutions, with each solution consisting of either 20% serum, 10% DMSO, and 70% media, or 95% serum and 5% DMSO.
  • The type of serum used varied across the samples, including autologous serum, commercially available pooled horse serum, and fetal bovine serum (FBS).
  • After the MSCs were thawed, the researchers assessed their cell survival, morphology, and growth kinetics by looking at total cell number, growth rates, the measure of colony-forming units, and changes in cell morphology during monolayer culture.

Results

  • No significant differences were found in post-thaw viability, cell count, morphology, or growth rates among the six solutions. Across all groups, post-thaw cell viabilities ranged between 80% to 90%.
  • Immediately after thawing, the majority of the MSCs stayed inactive while only a fraction proliferated. However, within 72 hours of thawing, approximately half of the cells started proliferating, particularly in the fourth generation.
  • The colony-forming unit assay, which helps determine the stem cells’ ability to divide and form colonies, showed a range of 72 to 115 colonies across all solutions.

Conclusions

  • The findings suggest that any of the serum sources could be used for short-term cryopreservation of equine MSCs; no solution stood out in terms of post-thaw cell viability, growth, or morphology.
  • Despite these results, the researchers suggest that for clinical applications, autologous serum may be preferred, potentially due to factors such as better control over variables and lower risk of reactions or infections.
  • The use of a lower DMSO concentration was also recommended, perhaps due to the compound’s potential toxicity at higher concentrations and the desire to minimize any possible risks associated with its use.

Cite This Article

APA
Mitchell A, Rivas KA, Smith R, Watts AE. (2015). Cryopreservation of equine mesenchymal stem cells in 95% autologous serum and 5% DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20 or 95% and DMSO at 10 or 5. Stem Cell Res Ther, 6, 231. https://doi.org/10.1186/s13287-015-0230-y

Publication

Stem cell research & therapy
ISSN: 1757-6512
NlmUniqueID: 101527581
Country: England
Language: English
Volume: 6
Pages: 231
PII: 231

Researcher Affiliations

Mitchell, Alexis
  • Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, 77843, USA. amitchell@cvm.tamu.edu.
Rivas, Kristen A
  • Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, 77843, USA. katwell8@uga.edu.
Smith, Roger
  • Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, 77843, USA. rosmith@cvm.tamu.edu.
Watts, Ashlee E
  • Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, 77843, USA. awatts@cvm.tamu.edu.

MeSH Terms

  • Animals
  • Bone Marrow Cells
  • Cattle
  • Cell Culture Techniques
  • Cell Survival
  • Cryopreservation
  • Cryoprotective Agents
  • Culture Media / chemistry
  • Dimethyl Sulfoxide
  • Horses
  • Mesenchymal Stem Cells
  • Serum
  • Species Specificity
  • Swine

References

This article includes 33 references
  1. Smith RK, Garvican ER, Fortier LA. The current 'state of play' of regenerative medicine in horses: what the horse can tell the human.. Regen Med 2014;9(5):673-85.
    doi: 10.2217/rme.14.42pubmed: 25372081google scholar: lookup
  2. Smith RK, Korda M, Blunn GW, Goodship AE. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment.. Equine Vet J 2003 Jan;35(1):99-102.
    doi: 10.2746/042516403775467388pubmed: 12553472google scholar: lookup
  3. Kim DH, Jamal N, Saragosa R, Loach D, Wright J, Gupta V, Kuruvilla J, Lipton JH, Minden M, Messner HA. Similar outcomes of cryopreserved allogeneic peripheral stem cell transplants (PBSCT) compared to fresh allografts.. Biol Blood Marrow Transplant 2007 Oct;13(10):1233-43.
    doi: 10.1016/j.bbmt.2007.07.003pubmed: 17889361google scholar: lookup
  4. Eckardt JR, Roodman GD, Boldt DH, Clark GM, Alvarez R, Page C, Gaskill H, LeMaistre CF. Comparison of engraftment and acute GVHD in patients undergoing cryopreserved or fresh allogeneic BMT.. Bone Marrow Transplant 1993 Feb;11(2):125-31.
    pubmed: 8435661
  5. Frey NV, Lazarus HM, Goldstein SC. Has allogeneic stem cell cryopreservation been given the 'cold shoulder'? An analysis of the pros and cons of using frozen versus fresh stem cell products in allogeneic stem cell transplantation.. Bone Marrow Transplant 2006 Sep;38(6):399-405.
    doi: 10.1038/sj.bmt.1705462pubmed: 16892075google scholar: lookup
  6. Parody R, Caballero D, Márquez-Malaver FJ, Vázquez L, Saldaña R, Madrigal MD, Calderón C, Carrillo E, Lopez-Corral L, Espigado I, Carmona M, López-Villar O, Pérez-Simón JA. To freeze or not to freeze peripheral blood stem cells prior to allogeneic transplantation from matched related donors.. Eur J Haematol 2013 Nov;91(5):448-55.
    doi: 10.1111/ejh.12140pubmed: 23710624google scholar: lookup
  7. Ikebe C, Suzuki K. Mesenchymal stem cells for regenerative therapy: optimization of cell preparation protocols.. Biomed Res Int 2014;2014:951512.
    doi: 10.1155/2014/951512pmc: PMC3912818pubmed: 24511552google scholar: lookup
  8. Garvican ER, Cree S, Bull L, Smith RK, Dudhia J. Viability of equine mesenchymal stem cells during transport and implantation.. Stem Cell Res Ther 2014 Aug 8;5(4):94.
    doi: 10.1186/scrt483pmc: PMC4247703pubmed: 25107289google scholar: lookup
  9. Ginis I, Grinblat B, Shirvan MH. Evaluation of bone marrow-derived mesenchymal stem cells after cryopreservation and hypothermic storage in clinically safe medium.. Tissue Eng Part C Methods 2012 Jun;18(6):453-63.
    doi: 10.1089/ten.tec.2011.0395pubmed: 22196031google scholar: lookup
  10. de Lima Prata K, de Santis GC, Orellana MD, Palma PV, Brassesco MS, Covas DT. Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions.. Cytotherapy 2012 Jul;14(6):694-700.
    doi: 10.3109/14653249.2012.677820pubmed: 22519634google scholar: lookup
  11. Adams MK, Goodrich LR, Rao S, Olea-Popelka F, Phillips N, Kisiday JD, McIlwraith CW. Equine bone marrow-derived mesenchymal stromal cells (BMDMSCs) from the ilium and sternum: are there differences?. Equine Vet J 2013 May;45(3):372-5.
    doi: 10.1111/j.2042-3306.2012.00646.xpmc: PMC3581704pubmed: 23009322google scholar: lookup
  12. Koch TG, Heerkens T, Thomsen PD, Betts DH. Isolation of mesenchymal stem cells from equine umbilical cord blood.. BMC Biotechnol 2007 May 30;7:26.
    doi: 10.1186/1472-6750-7-26pmc: PMC1904213pubmed: 17537254google scholar: lookup
  13. Radcliffe CH, Flaminio MJ, Fortier LA. Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations.. Stem Cells Dev 2010 Feb;19(2):269-82.
    doi: 10.1089/scd.2009.0091pmc: PMC3138180pubmed: 19604071google scholar: lookup
  14. Schnabel LV, Pezzanite LM, Antczak DF, Felippe MJ, Fortier LA. Equine bone marrow-derived mesenchymal stromal cells are heterogeneous in MHC class II expression and capable of inciting an immune response in vitro.. Stem Cell Res Ther 2014 Jan 24;5(1):13.
    doi: 10.1186/scrt402pmc: PMC4055004pubmed: 24461709google scholar: lookup
  15. English A, Jones EA, Corscadden D, Henshaw K, Chapman T, Emery P, McGonagle D. A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis.. Rheumatology (Oxford) 2007 Nov;46(11):1676-83.
    doi: 10.1093/rheumatology/kem217pubmed: 17901063google scholar: lookup
  16. Grogan SP, Barbero A, Winkelmann V, Rieser F, Fitzsimmons JS, O'Driscoll S, Martin I, Mainil-Varlet P. Visual histological grading system for the evaluation of in vitro-generated neocartilage.. Tissue Eng 2006 Aug;12(8):2141-9.
    doi: 10.1089/ten.2006.12.2141pubmed: 16968155google scholar: lookup
  17. Zhu Y, Ouyang Y, Chang Y, Luo C, Xu J, Zhang C, Huang W. Evaluation of the proliferation and differentiation behaviors of mesenchymal stem cells with partially converted borate glass containing different amounts of strontium in vitro.. Mol Med Rep 2013 Apr;7(4):1129-36.
    pubmed: 23446964doi: 10.3892/mmr.2013.1341google scholar: lookup
  18. Ivanov DP, Parker TL, Walker DA, Alexander C, Ashford MB, Gellert PR, Garnett MC. In vitro co-culture model of medulloblastoma and human neural stem cells for drug delivery assessment.. J Biotechnol 2015 Jul 10;205:3-13.
    doi: 10.1016/j.jbiotec.2015.01.002pubmed: 25592050google scholar: lookup
  19. Begum J, Day W, Henderson C, Purewal S, Cerveira J, Summers H, Rees P, Davies D, Filby A. A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution.. Cytometry A 2013 Dec;83(12):1085-95.
    pubmed: 24166880doi: 10.1002/cyto.a.22403google scholar: lookup
  20. Zolnierowicz J, Ambrozek-Latecka M, Kawiak J, Wasilewska D, Hoser G. Monitoring cell proliferation in vitro with different cellular fluorescent dyes.. Folia Histochem Cytobiol 2013;51(3):193-200.
    doi: 10.5603/FHC.2013.0027pubmed: 24203624google scholar: lookup
  21. Portalska KJ, Groen N, Krenning G, Georgi N, Mentink A, Harmsen MC, van Blitterswijk C, de Boer J. The effect of donor variation and senescence on endothelial differentiation of human mesenchymal stromal cells.. Tissue Eng Part A 2013 Nov;19(21-22):2318-29.
    doi: 10.1089/ten.tea.2012.0646pubmed: 23676150google scholar: lookup
  22. Haack-Sørensen M, Kastrup J. Cryopreservation and revival of mesenchymal stromal cells.. Methods Mol Biol 2011;698:161-74.
    doi: 10.1007/978-1-60761-999-4_13pubmed: 21431518google scholar: lookup
  23. Jung J, Moon N, Ahn JY, Oh EJ, Kim M, Cho CS, Shin JC, Oh IH. Mesenchymal stromal cells expanded in human allogenic cord blood serum display higher self-renewal and enhanced osteogenic potential.. Stem Cells Dev 2009 May;18(4):559-71.
    doi: 10.1089/scd.2008.0105pubmed: 18754716google scholar: lookup
  24. Asghar W, El Assal R, Shafiee H, Anchan RM, Demirci U. Preserving human cells for regenerative, reproductive, and transfusion medicine.. Biotechnol J 2014 Jul;9(7):895-903.
    doi: 10.1002/biot.201300074pmc: PMC4145864pubmed: 24995723google scholar: lookup
  25. LOVELOCK JE, BISHOP MW. Prevention of freezing damage to living cells by dimethyl sulphoxide.. Nature 1959 May 16;183(4672):1394-5.
    doi: 10.1038/1831394a0pubmed: 13657132google scholar: lookup
  26. Balint B, Ivanović Z, Petakov M, Taseski J, Jovcić G, Stojanović N, Milenković P. The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability.. Bone Marrow Transplant 1999 Mar;23(6):613-9.
    doi: 10.1038/sj.bmt.1701623pubmed: 10217193google scholar: lookup
  27. Berz D, McCormack EM, Winer ES, Colvin GA, Quesenberry PJ. Cryopreservation of hematopoietic stem cells.. Am J Hematol 2007 Jun;82(6):463-72.
    doi: 10.1002/ajh.20707pmc: PMC2075525pubmed: 17266054google scholar: lookup
  28. Weiner RS, Tobias JS, Yankee RA. The processing of human bone marrow for cryopreservation and reinfusion.. Biomedicine 1976;24(4):226-31.
    pubmed: 11006
  29. Dariolli R, Bassaneze V, Nakamuta JS, Omae SV, Campos LC, Krieger JE. Porcine adipose tissue-derived mesenchymal stem cells retain their proliferative characteristics, senescence, karyotype and plasticity after long-term cryopreservation.. PLoS One 2013;8(7):e67939.
    pmc: PMC3706624pubmed: 23874472doi: 10.1371/journal.pone.0067939google scholar: lookup
  30. Liu Y, Xu X, Ma X, Martin-Rendon E, Watt S, Cui Z. Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions.. Biotechnol Prog 2010 Nov-Dec;26(6):1635-43.
    pubmed: 20572296doi: 10.1002/btpr.464google scholar: lookup
  31. Liu Y, Xu X, Ma XH, Liu J, Cui ZF. Effect of various freezing solutions on cryopreservation of mesenchymal stem cells from different animal species.. Cryo Letters 2011 Sep-Oct;32(5):425-35.
    pubmed: 22020465
  32. Luetzkendorf J, Nerger K, Hering J, Moegel A, Hoffmann K, Hoefers C, Mueller-Tidow C, Mueller LP. Cryopreservation does not alter main characteristics of Good Manufacturing Process-grade human multipotent mesenchymal stromal cells including immunomodulating potential and lack of malignant transformation.. Cytotherapy 2015 Feb;17(2):186-98.
    pubmed: 25593077doi: 10.1016/j.jcyt.2014.10.018google scholar: lookup
  33. Tateishi K, Ando W, Higuchi C, Hart DA, Hashimoto J, Nakata K, Yoshikawa H, Nakamura N. Comparison of human serum with fetal bovine serum for expansion and differentiation of human synovial MSC: potential feasibility for clinical applications.. Cell Transplant 2008;17(5):549-57.
    pubmed: 18714674doi: 10.3727/096368908785096024google scholar: lookup

Citations

This article has been cited 15 times.
  1. Rowland AL, Burns ME, Levine GJ, Watts AE. Preparation Technique Affects Recipient Immune Targeting of Autologous Mesenchymal Stem Cells. Front Vet Sci 2021;8:724041.
    doi: 10.3389/fvets.2021.724041pubmed: 34595230google scholar: lookup
  2. Tonarova P, Lochovska K, Pytlik R, Hubalek Kalbacova M. The Impact of Various Culture Conditions on Human Mesenchymal Stromal Cells Metabolism. Stem Cells Int 2021;2021:6659244.
    doi: 10.1155/2021/6659244pubmed: 33727935google scholar: lookup
  3. Rowland AL, Miller D, Berglund A, Schnabel LV, Levine GJ, Antczak DF, Watts AE. Cross-matching of allogeneic mesenchymal stromal cells eliminates recipient immune targeting. Stem Cells Transl Med 2021 May;10(5):694-710.
    doi: 10.1002/sctm.20-0435pubmed: 33369287google scholar: lookup
  4. Bahsoun S, Coopman K, Akam EC. Quantitative assessment of the impact of cryopreservation on human bone marrow-derived mesenchymal stem cells: up to 24 h post-thaw and beyond. Stem Cell Res Ther 2020 Dec 14;11(1):540.
    doi: 10.1186/s13287-020-02054-2pubmed: 33317625google scholar: lookup
  5. Ge Z, Tang H, Chen W, Wang Y, Yuan C, Tao X, Zhou B, Tang K. Downregulation of type I collagen expression in the Achilles tendon by dexamethasone: a controlled laboratory study. J Orthop Surg Res 2020 Feb 24;15(1):70.
    doi: 10.1186/s13018-020-01602-zpubmed: 32093733google scholar: lookup
  6. Enciso N, Amiel J, Pando J, Enciso J. Multidose intramuscular allogeneic adipose stem cells decrease the severity of canine atopic dermatitis: A pilot study. Vet World 2019 Nov;12(11):1747-1754.
    doi: 10.14202/vetworld.2019.1747-1754pubmed: 32025111google scholar: lookup
  7. Bahsoun S, Coopman K, Akam EC. The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review. J Transl Med 2019 Nov 29;17(1):397.
    doi: 10.1186/s12967-019-02136-7pubmed: 31783866google scholar: lookup
  8. Grady ST, Britton L, Hinrichs K, Nixon AJ, Watts AE. Persistence of fluorescent nanoparticle-labelled bone marrow mesenchymal stem cells in vitro and after intra-articular injection. J Tissue Eng Regen Med 2019 Feb;13(2):191-202.
    doi: 10.1002/term.2781pubmed: 30536848google scholar: lookup
  9. Grady ST, Watts AE, Thompson JA, Penedo MCT, Konganti K, Hinrichs K. Effect of intra-ovarian injection of mesenchymal stem cells in aged mares. J Assist Reprod Genet 2019 Mar;36(3):543-556.
    doi: 10.1007/s10815-018-1371-6pubmed: 30470961google scholar: lookup
  10. Al-Farha AA, Khazandi M, Hemmatzadeh F, Jozani R, Tearle R, Hoare A, Petrovski K. Evaluation of three cryoprotectants used with bovine milk affected with Mycoplasma bovis in different freezing conditions. BMC Res Notes 2018 Apr 2;11(1):216.
    doi: 10.1186/s13104-018-3325-6pubmed: 29609634google scholar: lookup
  11. Duan W, Lopez MJ, Hicok K. Adult multipotent stromal cell cryopreservation: Pluses and pitfalls. Vet Surg 2018 Jan;47(1):19-29.
    doi: 10.1111/vsu.12730pubmed: 29023790google scholar: lookup
  12. Yaneselli KM, Kuhl CP, Terraciano PB, de Oliveira FS, Pizzato SB, Pazza K, Magrisso AB, Torman V, Rial A, Moreno M, Llambí S, Cirne-Lima E, Maisonnave J. Comparison of the characteristics of canine adipose tissue-derived mesenchymal stem cells extracted from different sites and at different passage numbers. J Vet Sci 2018 Jan 31;19(1):13-20.
    doi: 10.4142/jvs.2018.19.1.13pubmed: 28693305google scholar: lookup
  13. Joswig AJ, Mitchell A, Cummings KJ, Levine GJ, Gregory CA, Smith R 3rd, Watts AE. Repeated intra-articular injection of allogeneic mesenchymal stem cells causes an adverse response compared to autologous cells in the equine model. Stem Cell Res Ther 2017 Feb 28;8(1):42.
    doi: 10.1186/s13287-017-0503-8pubmed: 28241885google scholar: lookup
  14. Espina M, Jülke H, Brehm W, Ribitsch I, Winter K, Delling U. Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses. PeerJ 2016;4:e1773.
    doi: 10.7717/peerj.1773pubmed: 27019778google scholar: lookup
  15. Nascimento C, Saraiva MVA, Pereira VM, de Brito DCC, de Aguiar FLN, Alves BG, Roballo KCS, de Figueiredo JR, Ambrósio CE, Rodrigues APR. Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration. Braz J Vet Med 2023;45:e002523.
    doi: 10.29374/2527-2179.bjvm002523pubmed: 38162818google scholar: lookup
Subscribe to our newsletter
Join 99,824 horse owners like you who receive our email updates on horse health and nutrition.