Abstract
Umbilical cord blood (UCB) is an extremely attractive source of stem cells for the treatment of various benign and malignant hematological and non-hematological disorders. To facilitate the preservation of these stem cells, 10 % dimethylsulfoxide (DMSO) is widely used as cryoprotectant in cord blood banks. But it is found to be toxic at this concentration with the result of serious side effects in recipients after infusion of DMSO-cryopreserved cells. Evaluation of viability and functionality of cryopreserved hematopoietic stem cells is needed with either inclusion of nontoxic additives alone or with reduced DMSO concentration. We assessed the post thawing viability of UCB stem cells in the freezing medium containing disaccharides (sucrose or trehalose) alone and in combination with reduced amount i.e. 2 % DMSO by trypan blue staining. The functionally active progenitor cells content of the optimized media was then evaluated and compared with 5% DMSO by a colony forming unit assay using methylcellulose based media. The freezing solution containing 0.2 M trehalose with 2 % DMSO came out to be superior in the evaluation of viability and generation of hematopoietic colonies of erythroid and myeloid lineage than 5 % DMSO alone. While the percentage of viability was lower than 2 % DMSO, as observed in the medium containing 0.2 M trehalose or sucrose alone, with poor outcome of generation of myeloid lineage based colonies. Our study results suggest that trehalose (0.2M) with the inclusion of reduced concentration of DMSO(2%) can better replace 5%DMSO rather than complete removal of DMSO from the freezing medium.
Similar content being viewed by others
References
Brooke G, Rossetti T, Pelekanos R, Ilic N, Murray P, Hancock S et al (2009) Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials. Br J Haematol 144:571–579
Thirumala S, Zvonic S, Floyd E, Gimble JM, Devireddy RV (2005) Effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells. Biotechnol Prog 21:1511–1524
Goh BC, Thirumala S, Kilroy G, Devireddy RV, Gimble JM (2007) Cryopreservation characteristics of adipose-derived stem cells: maintenance of differentiation potential and viability. J Tissue Eng Regen Med 1:322–324
Liu G, Zhou H, Li Y, Li G, Cui L, Liu W et al (2008) Evaluation of the viability and osteogenic differentiation of cryopreserved human adipose-derived stem cells. Cryobiology 57:18–24
Woods EJ, Benson JD, Agca Y, Critser JK (2004) Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48:146–156
Hunt CJ, Armitage SE, Pegg DE (2003) Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing. Cryobiology 46:76–87
Donaldson C, Armitage WJ, Denning-Kendall PA, Nicol AJ, Bradley BA, Hows JM (1996) Optimal cryopreservation of human umbilical cord blood. Bone Marrow Transplant 18:725–731
Alessandrino P, Bernasconi P, Caldera D, Colombo A, Bonfichi M, Malcovati L et al (1999) Adverse events occurring during bone marrow or peripheral blood progenitor cell infusion: analysis of 126 cases. Bone Marrow Transplant 23:533–537
Benekli M, Anderson B, Wentling D, Bernstein S, Czuczman M, McCarthy P (2000) Severe respiratory depression after dimethylsulphoxide-containing autologous stem cell infusion in a patient with AL amyloidosis. Bone Marrow Transplant 25:1299–1301
Hoyt R, Szer J, Grigg A (2000) Neurological events associated with the infusion of cryopreserved bone marrow and/or peripheral blood progenitor cells. Bone Marrow Transplant 25:1285–1287
Zenhausern R, Tobler A, Leoncini L, Hess OM, Ferrari P (2000) Fatal cardiac arrhythmia after infusion of dimethyl sulfoxide-cryopreserved hematopoietic stem cells in a patient with severe primary cardiac amyloidosis and end-stage renal failure. Ann Hematol 79:523–526
Haines AH (2006) Non-equivalence of d- and l-trehalose in stabilizing alkaline phosphatase against freeze-drying and thermal stress. Is chiral recognition involved? Org Biomol Chem 4:702–706
Carpenter JF, Crowe JH (1989) An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry 28:3916–3922
Jovanovic N, Bouchard A, Hofland GW, Witkamp GJ, Crommelin DJ, Jiskoot W (2006) Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying. Eur J Pharm Sci 27:336–345
Patist A, Zoerb H (2005) Preservation mechanisms of trehalose in food and biosystems. Colloids Surf B 40:107–113
Kravchenko LP, Petrenko AYu, Somov AYu et al (2001) Respiratory activity of isolated rat hepatocytes following cold storage and subsequent rewarming: a comparison of sucrose-based and University of Wisconsin solutions. Cryobiology 42:218–221
Kravchenko LP, Semenchenko OA, Fuller BJ (2004) ATP level in whole rat liver during cold hypoxia and subsequent rewarming. Ukr Biochem J 76:136–138
Petrenko AYu, Grischuk VP, Roslyakov AD et al (1992) Survival metabolic activity and transport of potassium ions of rat hepatocytes after rapid freeze–thawing under protection of dimethylsulfoxide and separation in Percoll density Gradient. Cryo Lett 13:87–98
Sola-Penna M, Ferreira-Pereira A, Lemos AP, Meyer-Fernandes JR (1997) Carbohydrate protection of enzyme structure and function against guanidinium chloride treatment depends on the nature of carbohydrate and enzyme. Eur J Biochem 248:24–29
Rudolph AS, Crowe JH, Crowe LM (1986) Effects of three stabilizing agents—proline, betaine, and trehalose on membrane phospholipids. Arch Biochem Biophys 245:134–143
Limaye LS, Kale VP (2001) Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium. J Hematother Stem Cell Res 10:709–718
Zhang XB, Li K, Yau KH et al (2003) Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long-term culture-initiating cells, and nonobese diabetic SCID repopulating cells. Transfusion 43:265–272
Buchanan SS, Gross SA, Acker JP, Toner M, Carpenter JF, Pyatt DW (2004) Cryopreservation of stem cells using trehalose: evaluation of the method using a human hematopoietic cell line. Stem Cells Dev 13:295–305
Jain NK, Roy I (2008) Role of trehalose in moisture induced aggregation of bovine serum albumin. Eur J Pharm Biopharm 69:824–834
Han Y, Jin BS, Lee SB, Sohn Y, Joung JW, Lee JH (2007) Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage. Arch Pharm Res 30:1124–1131
Rodrigues JP, Paraguassú-Braga FH, Carvalho L, Abdelhay E, Bouzas LF, Porto LC (2008) Evaluation of trehalose and sucrose as cryoprotectants for hematopoietic stem cells of umbilical cord blood. Cryobiology 56:144–151
Sasnoor LM, Kale VP, Limaye LS (2003) Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells. J Hematother Stem Cell Res 12(5):553–564
Sasnoor LM, Kale VP, Limaye LS (2005) A combination of catalase and trehalose as additives to conventional freezing medium results in improved cryoprotection of human hematopoietic cells with reference to in vitro migration and adhesion properties. Transfusion 45(4):622–633
Fuller B, Paynter S (2004) Fundamentals of cryobiology in reproductive medicine. Reprod Biomed Online 9:680–691
Mantri S, Mohapatra PC (2013) Evaluation and optimization of storage conditions of cord blood stem cells. Biomedicine 33(2):215–223
Mahmut N, Katayama Y, Takenaka K et al (1999) Analysis of circulating hematopoietic progenitor cells after peripheral blood stem cell transplantation. Int J Hematol 69:36–42
Sputtek A, Jetter S, Hummel K, Kuhnl P (1997) Cryopreservation of peripheral blood progenitor cells: characteristics of suitable techniques. Beitr Infusionsther Transfusionsmed 34:79–83
Fahy GM, Levy DI, Ali SE (1987) Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology 24:196–213
Kim SI, Choi HK, Son JS et al (2001) Cryopreservation of Taxus chinensis suspension cell cultures. Cryo Lett 22:43–50
Leslie SB, Israeli E, Lighthart B et al (1995) Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol 61:3592–3597
Yokomise H, Inui K, Wada H et al (1995) Reliable cryopreservation of trachea for one month in a new trehalose solution. J Thorac Cardiovasc Surg 110:382–385
Scheinko¨ nig C, Kappicht S, Kolb H-J, Schleuning M (2004) Adoption of long-term cultures to evaluate the cryoprotective potential of trehalose for freezing hematopoietic stem cells. Bone Marrow Transplant 34:531–536
Acknowledgments
This study was supported by the grant from Department of Science & Technology (DST), Govt. of India.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mantri, S., Kanungo, S. & Mohapatra, P.C. Cryoprotective Effect of Disaccharides on Cord Blood Stem Cells with Minimal Use of DMSO. Indian J Hematol Blood Transfus 31, 206–212 (2015). https://doi.org/10.1007/s12288-014-0352-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12288-014-0352-x