The effects of cryopreservation on human dental pulp-derived mesenchymal stem cells

  • Tomlin, Allison ;
  • Sanders, Michael B ;
  • Kingsley, Karl
  • Received : 2016.04.01
  • Accepted : 2016.05.31
  • Published : 2016.06.25


The purpose of this study is to evaluate the effects of cryopreservation on dental pulp-derived stem cells (DPSC) viability over a period of three years. Dental pulp-derived stem cells were isolated and cultured from thirty-one healthy teeth. DPSC isolates were assessed for doubling-time and baseline viability prior to cryopreservation and were assessed again at three time points; one week (T1), 18 months (T2), and 36 months (T3). DPSC can be grouped based on their observed doubling times; slow (sDT), intermediate (iDT), and rapid (rDT). Viability results demonstrated all three types of DPSC isolates (sDT, iDT and rDT) exhibit time-dependent reductions in viability following cryopreservation, with the greatest reduction observed among sDT-DPSCs and the smallest observed among the rDT-DPSC isolates. Cryopreserved DPSCs demonstrate time-dependent reductions in cellular viability. Although reductions in viability were smallest at the initial time point (T1) and greatest at the final time point (T3), these changes were markedly different among DPSC isolates with similar doubling times (DTs). Furthermore, the analysis of various DPSC biomarkers - including both intracellular and cell surface markers, revealed differential mRNA expression. More specifically, the relative high expression of Sox-2 was only found only among the rDT isolates, which was associated with the smallest reduction in viability over time. The expression of Oct4 and NANOG were also higher among rDT isolates, however, expression was comparatively lower among the sDT isolates that had the highest reduction in cellular viability over the course of this study. These data may suggest that some biomarkers, including Sox-2, Oct4 and NANOG may have some potential for use as biomarkers that may be associated with either higher or lower cellular viability over long-term storage applications although more research will be needed to confirm these findings.


cryopreservation;human dental pulp-derived stem cells;effect


  1. Alleman, M., Low, E., Truong, K., Huang, E., Hill, C.K., Chen, T.Y., ... and Kingsley, K. (2013), "Dental pulp-derived stem cells (DPSC) differentiation in vitro into odontoblast and neuronal progenitors during cell passaging is associated with alterations in cell survival and viability", Int. J. Medicine Biomed. Res., 2(2), 133-141.
  2. Burnett, A., Kumar, R., Westphal, J.D. and Kingsley, K. (2015), "Dichloroacetate (DCA) Promotes a De-Differentiated Phenotype in Dental Pulp-Derived Stem Cells in vitro", Int. J. Biologic. Sci. Appl., 2(3), 25-32.
  3. Conde, M.C.M., Chisini, L.A., Demarco, F.F., Nor, J.E., Casagrande, L. and Tarquinio, S.B.C. (2015), "Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature", Int. Endod. J.,
  4. Ducret, M., Fabre, H., Farges, J.C., Degoul, O., Atzeni, G., McGuckin, C., ... and Perrier-Groult, E. (2015), "Production of human dental pulp cells with a medicinal manufacturing approach", J. Endod., 41(9), 1492-1499.
  5. Ferro, F., Spelat, R. and Baheney, C.S. (2014), "Dental pulp stem cell (DPSC) isolation, characterization, and differentiation", Stem Cells and Tissue Repair: Methods and Protocols, 91-115.
  6. Gioventu, S., Andriolo, G., Bonino, F., Frasca, S., Lazzari, L., Montelatici, E., ... and Rebulla, P. (2012), "A novel method for banking dental pulp stem cells", Transfus. Apher. Sci., 47(2), 199-206.
  7. Gronthos, S., Arthur, A., Bartold, P.M. and Shi, S. (2011), "A method to isolate and culture expand human dental pulp stem cells", Mesenchymal Stem Cell Assays Appl., 107-121.
  8. Hays WL: Inferences about population means. In: Statistics (5th edition). International Thomson Publishing 1994. 311-342.
  9. Huang, G.J., Gronthos, S. and Shi, S. (2009), "Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine", J. Dental Res., 88(9), 792-806.
  10. Hung, E., Lee, S., Fitzgerald, B., Hill, C.K. and Kingsley, K. (2013), "Dental pulp-derived stem cell (DPSC) survival and viability may correlate with specific patient demographics", Forum for Dental Student Research and Innovation (FDSRI), 1(3), 14-21.
  11. Kumar, A., Bhattacharyya, S. and Rattan, V. (2015), "Effect of uncontrolled freezing on biological characteristics of human dental pulp stem cells", Cell tissue bank., 16(4), 513-522.
  12. Lee, S.Y., Huang, G.W., Shiung, J.N., Huang, Y.H., Jeng, J.H., Kuo, T.F., ... and Yang, W.C. (2012), "Magnetic cryopreservation for dental pulp stem cells", Cells Tissues Organs, 196(1), 23-33.
  13. Lindemann, D., Werle, S.B., Steffens, D., Garcia-Godoy, F., Pranke, P. and Casagrande, L. (2014), "Effects of cryopreservation on the characteristics of dental pulp stem cells of intact deciduous teeth", Arch. Oral Biology, 59(9), 970-976.
  14. Loveland, K., Young, A., Khadiv, M., Culpepper, M. and Kingsley, K. (2014), "Dental Pulp Stem Cell (DPSC) pluripotency enhanced by Transforming Growth Factor (TGF-1) in Vitro may be inhibited by differentiation-inducing factors Laminin-5 and Dexamethasone", Int. J. Biologic. Sci. Appl., 1(3), 55-61.
  15. Perry, B.C., Zhou, D., Wu, X., Yang, F.C., Byers, M.A., Chu, T.M.G., ... and Goebel, W.S. (2008), "Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use", Tissue Eng. Part C: Method., 14(2), 149-156.
  16. Pisciotta, A., Carnevale, G., Meloni, S., Riccio, M., De Biasi, S., Gibellini, L., ... and De Pol, A. (2015), "Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations", BMC Development. Biology, 15(1), 1.
  17. Potdar, P.D. and Jethmalani, Y.D. (2015), "Human dental pulp stem cells: Applications in future regenerative medicine", World J. Stem Cell., 7(5), 839.
  18. Ravindran, S. and George, A. (2015), "Biomimetic extracellular matrix mediated somatic stem cell differentiation: applications in dental pulp tissue regeneration", Front. Phys., 6, 118.
  19. Saito, M.T., Silverio, K.G., Casati, M.Z., Sallum, E.A. and Nociti Jr, F.H. (2015), "Tooth-derived stem cells: update and perspectives", World J. Stem Cell., 7(2), 399-407.
  20. Tatullo, M., Marrelli, M., Shakesheff, K.M. and White, L.J. (2015), "Dental pulp stem cells: function, isolation and applications in regenerative medicine", J. Tissue Eng. Regenerat. Med., 9(11), 1205-1216.
  21. Woods, E.J., Perry, B.C., Hockema, J.J., Larson, L., Zhou, D. and Goebel, W.S. (2009), "Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use", Cryobiology, 59(2), 150-157.
  22. Xiao, L. and Nasu, M. (2014), "From regenerative dentistry to regenerative medicine: progress, challenges, and potential applications of oral stem cells", Stem Cells Clon., 7(Dec), 89-99.
  23. Zhurova, M., Woods, E.J. and Acker, J.P. (2010), "Intracellular ice formation in confluent monolayers of human dental stem cells and membrane damage", Cryobiology, 61(1), 133-141.