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Stem cells cryopreservation

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Stefy kthe virtualdentalcongress

Stefy kthe virtualdentalcongress

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  • 1. Human Dental Pulp Stem Cells Cryopreservation Stefanny Romero Oyuela, Katherin Córdoba Pérez, Sandra Perdomo, Juan Carlos Munévar Niño. stephiromero@hotmail.comInstituto Unidad de Investigación Básica Oral. U.I.B.O. Universidad El Bosque, Bogotá, Colombia.
  • 2. INTRODUCTION Due to the high prevalence of NEWcraniofacial, dental THERAPEUTICAL and periodontal APPROACHES pathologies
  • 3. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Booyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002; 81 (8): 531-5Iohara K. Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. J Dent Res. 2004; 83(8):590-5.
  • 4. INTRODUCTION The human dental pulp Stem cells (DPSCs) are fundamental in regenerative dentistry & medicine Optimal DSC cryopreservation METHODS for later clinical use. U.I.B.O. There are several challenges in relation to the quality and safety ofhuman dental stem cells cryopreservation, particularly those related tothe conservation of these ex-vivo cells at extremely low temperatures.Gronthos et al/ 2000 Shi et al/ 2001
  • 5. OBJECTIVE To evaluate the effect of twocryopreservation methods on theviability and phenotype of hDPSCs
  • 6. Selection SampleAn in vitro experimental study criteria Informed consent approved by CD105+ the Institutional Review Board. 18- 31 years cells 48 healthy tooth Erupted premolars and molars Systemically healthy patients
  • 7. 1. Dental pulp sample transport to 2. Enzymatic Dissociationthe cell culture laboratory (UIBO) Enzymatic solution: 3 mg/ml collagenase Dissection & dental pulp explants and 4 mg/ml dispase in DMEM 4. Cell Isolation 3. Viability evaluation Tripan blue / Neubauer chamber By CD 105+ magnetic beads / MILTENYI MiniMACS
  • 8. 5. In vitro CD105+ expansion The in vitro expansion were done in 27 wells, for each protocol for 6. CRYOPRESERVATION cryopreservation and the control group Protocol No.1 Protocol No.2 • Incubation at 37 C Papaccio et Kamath • Humid atmosphere total of 81 wells al/2006 Fischer • Cell culture confluence (>70 %), Scientific 7. STATISTICAL ANALYSIS Kruskal-Wallis ANOVA TamhanePapaccio G, Graziano A, d´Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G. Long-term Cryopreservation of dental pulp stem cells (SBP-DPSCs) andtheir differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol. 2006 Aug; 208(2):319-25. Kamath A. Human Mesenchymal Stem Cell Protocol: Cryopreservation. SC Protocol Sheet: 00007. Cellular Engineering Technologies, Inc. Thermo Fisher Scientific Inc. 2007
  • 9. Results Phase contrast microscopy analysis Human dental pulp samples In vitro human dental pulp stem cells.In vitro dental pulp stem cells morphology. DPSCs Colony forming Unit CFU
  • 10. DPSCs viability by Flow cytometry Cryopreservation method 1: Papaccio et al/ 2006 A B 56.2 % DPSCs viability at 24 hours. 55.7 % DPSCs viability at 7 days.Papaccio G, Graziano A, d´Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G. Long-term Cryopreservation of dental pulp stem cells(SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol. 2006 Aug; 208(2):319-25.
  • 11. DPSCs phenotype Cryopreservation method 1: Papaccio et al/ 2006 A B C D E F A. CD105+/CD34- cells at 24 hours B. CD105+/CD45- cells at 24-hours C. CD34-/CD45- cells at 24 hours. D. CD105+/CD34- cells at 7 days E. CD105+/CD45- cells at 7 days, F. CD34-/CD45- cells at 7 days.
  • 12. DPSCs viability by Flow cytometry Cryopreservation method 2: Kamath (Fischer Scientific)/ 2007 A B 68.4% 61.2% 68,4 % DPSCs viability at 24 hours. 61,2 % DPSCs viability at 7 days.
  • 13. DPSCs phenotype Cryopreservation method 2: Kamath (Fischer Scientific)/ 2007 A B C D E F A. CD105+/CD34- cells at 24 hours B. CD105+/CD45- cells at 24-hours C. CD34-/CD45- cells at 24 hours. D. CD105+/CD34- cells at 7 days E. CD105+/CD45- cells at 7 days, F. CD34-/CD45- cells at 7 days.
  • 14. DPSCs viability Cryopreservation method 1 / Cryopreservation method 2
  • 15. Results Statistical Analysis DPSCs viability post cryopreservation between the 2 methods
  • 16. DPSCs phenotype Cryopreservation method 1 / Cryopreservation method 2
  • 17. Results Statistical Analysis DPSCs phenotype post cryopreservation between the 2 methods
  • 18. Recent studies describe methods for DPSCs characterization, isolation and cell culture Gronthos et al/2000. Miura et al/2003. Iohara et al/ 2006. PNAS PNAS. Stem Cells. Papaccio et al/2006. Perry et al/2008. Woods et al/ 2009. J Cell Physiol. Tissue Eng Methods. Cryobiology Although they do not analyze factors which may be decisive in the effectiveness of cryopreservation protocols, as suggested by the results of this study.Woods EJ, Perry BC, Hockema J, Larson L, Zhou D, Goebe W, et al. Optimized cryopreservation method for human dentalpulp-derived stem cells and their tissues. Elseiver. Cryobiology. 2009; 59(2): 150-157.
  • 19. There is a significant and inverse correlation between The ideal teeth: third molars and deciduous teeth. the sample transport and the number of CD 105+ DPSC´s It is reported a greater There is a mild and differentiation potential of indirect relationship mesenchymal stem cells in between the patients age connective tissues of younger and the number of patients CD105+ DPSCs isolated. (Gronthos et al/ 2002).Woods EJ, Perry BC, Hockema J, Larson L, Zhou D, Goebe W, et al. Optimized cryopreservation method for human dentalpulp-derived stem cells and their tissues. Elseiver. Cryobiology. 2009; 59(2): 150-157.
  • 20. Although the results are not It is essential to evaluate theconclusive due to the reduced effect of two cryopreservation sample size, they show methods for longer times on the important trends for an viability and phenotype of optimal protocol that must mesenchymal stem cells of pulpal be taken into account for an origin. effective DPSCs isolation. The method currently investigated and used is the cryopreservation which consist in freezing samples in order to reduce their metabolic activity and maintain low temperatures for long periods, while preserving its viability.
  • 21. 1. Thirumala S, Goebel WS, Woods EJ. Clinical grade adult stem cell banking. Organogenesis 2009; 5(3): 143-154.2. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng. 2001; 7(2):211-28.3. Brooke G, Rossetti T, Pelekanos R, Ilic N, Murray P, Hancock S, et al. Manufacturing of human placentaderivedmesenchymal stem cells for clinical trials. British Journal Haematology 2009; 144 (4):571-9.4. Laino G, Carinci F, Graziano A, Papaccio G. In vitro bone production using Stem cells derived from human dentalpulp. J Craniofac Surg. 2006; 17(3):511-515.5. Miura M, Gronthos S, Zhao M, Lu B, Fisher L, Robey P, et al. SHED: stem cells from human exfoliated deciduousteeth. 2003; 100 (10):5807–5812.6. Graziano A, d’Aquino R, Cusella-De Angelis MG, Laino G, Piattelli A. Concave pit-containing scaffold surfacesimprove stem cell-derived osteoblast performance and lead to significant bone tissue formation. PLoS One. 2007; 2(6):e496.7. Troyer DL, Weiss ML. Whartons jelly-derived cells are a primitive stromal cell population. Stem Cells. 2008; 26(3):591-5998. Iohara K. Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein2. J Dent Res. 2004; 83(8):590-5.9. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stemcells. J Dent Res. 2002; 81 (8): 531-5.1-442.10. Woods EJ, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. Cryobiology.2004; 48(2):146-56.12. Temmerman L, Beele H, Dermaut L, Van Maele G, De Pauw G. Influence of cryopreservation on the pulpal tissue ofimmature third molars in vitro. Cell tissue bank. 2010; 11(3): 281-289.13. Papaccio G, Graziano A, d Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G. Long-termCryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissuerepair. J Cell Physiol. 2006 Aug; 208(2):319-25.