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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.com
Instituto Unidad de Investigación Básica Oral. U.I.B.O.
Universidad El Bosque, Bogotá, Colombia.

2. INTRODUCTION
Due to the high
prevalence of
NEW
craniofacial, 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): 531-5
Iohara 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 of
human dental stem cells cryopreservation, particularly those related to
the 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 two
cryopreservation methods on the
viability and phenotype of hDPSCs

6. Selection
Sample
An 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 Dissociation
the 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
Tamhane
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.
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 dental
pulp-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 patient's age connective tissues of younger
and the number of patients
CD105+ DPSC's isolated. (Gronthos et al/ 2002).
Woods EJ, Perry BC, Hockema J, Larson L, Zhou D, Goebe W, et al. Optimized cryopreservation method for human dental
pulp-derived stem cells and their tissues. Elseiver. Cryobiology. 2009; 59(2): 150-157.

20. Although the results are not It is essential to evaluate the
conclusive 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.

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3. Brooke G, Rossetti T, Pelekanos R, Ilic N, Murray P, Hancock S, et al. Manufacturing of human placentaderived
mesenchymal 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 dental
pulp. 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 deciduous
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6. Graziano A, d’Aquino R, Cusella-De Angelis MG, Laino G, Piattelli A. Concave pit-containing scaffold surfaces
improve stem cell-derived osteoblast performance and lead to significant bone tissue formation. PLoS One. 2007; 2(6):
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591-599
8. Iohara K. Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein
2. 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 stem
cells. J Dent Res. 2002; 81 (8): 531-5.
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10. Woods EJ, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. Cryobiology.
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12. Temmerman L, Beele H, Dermaut L, Van Maele G, De Pauw G. Influence of cryopreservation on the pulpal tissue of
immature 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-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.