Poster #566
In vitro ISOLATION OF HUMAN DENTAL PULP STEM CELLS N. GUTIERREZ, J. MUNÉVAR, M. TAMAYO, L. RODRIGUEZ, C. VELANDIA, A. GÓMEZ, D. DORTA, A. MIRANDA, and J. FORERO, Unidad de Investigación Básica Oral U.I.B.O, Universidad El Bosque, Bogotá, Colombia nicolegutierrez@hotmail.com
1. INTRODUCTION
3. MATERIALS AND METHODS
Due to the high prevalence of dental and periodontal pathologies that cause irreversible damage to teeth and maxillary structures, it is necessary to approach new therapeutical strategies. The human dental pulp Stem cells (DPSCs), basic in the mechanisms of tissue development and regeneration (Gronthos et al/ 2000 Shi et al/ 2001) are fundamental in regenerative medicine and dentistry; therefore it is important to find an isolation method for optimal cryopreservation for later clinical use. However, there are several challenges in relation to the quality and safety in clinical applications of adult stem cells, particularly those related to the conservation of these ex-vivo cells at extremely low temperatures.
2. OBJECTIVE To establish the isolation method for future cryopreservation of DPSCs determining factors as age, tooth type, collection and processing times that can influence their quality and quantity.
4. CD105+ cell culture
4.RESULTS
The analysis demonstrate an inverse, moderate and statistically significant association between the collection and the processing times of the sample with the number of cells isolated (p=0,06 and 0.09 respectively). Other associations did not show to be statistically significant. Pool Sample
Patient age *
1 2 3 4 5 6 7 8 9 10 Total ¥ and/ ∞ 27 or average * years of age ** hours
28 24 28 27 29 29 31 30 18 26 +3.74
∞
Tooth 3rd Molar premolar Erupted Included 2 1 2 2 2 2 2 2 2 4 2 12
¥
¥
¥
9
2
Obtention time of the samples ** 4 2 3 2 1 2 12 14 15 2 5.7 +5.9
Processing time of the samples ** 5 5 5 5 5 5 4 4 4 4
∞
1A
Fig 1. Fibroblast-like morphology of DPSCs confluent after 18 days of observation in primary culture. Phase contrast microscope. (1A): 40x (1B): 20X
∞
4.6+0.71
2A
Table 1. Detailed description of the determining factors evaluated.
POOL sample
N° of pulp cells/ml isolated with MEDIMACHINE 280.000 410.000 255.000 250.000 425.000 225.000 260.000 220.000 115.000 240.000
1B
Fig 2. (2A): Positive control mesenchymal stem cells from Whaton’s Jelly of Umbilical Cord. (2B): Negative control human fibroblasts (40X)
N° of stem cells/ ml % CD 105 N° of wells for Isolated with + POOL sample MILTENYI 45.000 16 9 90.000 21 18 40.000 15 8 30.000 12 6 50.000 12 10 25.000 11 5 52.000 20 11 30.000 14 6 20.000 17 4 40.000 17 8
1 2 3 4 5 6 7 8 9 10 Average 268.000+90.590 cels/ml 35.285+12.338 cels/ml 16%+0.03 – DS
8.5+4
Table 2. Detailed description of expected results
2B
Collection time of samples
Processing time of cultures
Fig 3. (A): Correlation between the collection time of the samples (h) and the number of pulp cells obtained after mechanical disgregation with Medimachine. (B): Correlation between processing time of the samples (h) and the number of pulp cells obtained after mechanical disgregation with Medimachine.
DPSCs VIABILITY & PHENOTYPE AFTER CRYOPRESERVATION
Fig 4. Method N°1. Negative control without antibodies
Fig 5. DPSCs viability and phenotype CD105+/CD34-/CD45– after 24 hours in cryopreservation. Method N°1
Fig 7. DPSCs Viability and phenotype CD105+/CD34-/CD45– after 7 days in cryopreservation. Method N°1
Fig 6. DPSCs viability and phenotype CD105+/CD34-/CD45– after 24 hours in cryopreservation. Method N° 2
Fig 8. DPSCs viability and phenotype CD105+/CD34-/CD45– after 7 days in cryopreservation. Method N°2
100%
99%
DPSCs Phenotype Method 1
1 day
7 days
CD105+/CD34CD105+/CD45CD34-/CD45-
99% 95,40% 96,60%
100% 98,10% 98,80%
98%
97%
DPSCs phenotype 1 day
DPSCs phenotype 7 days 96%
95%
DPSCs Phenotype Method 2 CD105+/CD34CD105+/CD45CD34-/CD45-
94%
1 day 95% 93,00%
7 days 98% 96,30%
99,30%
99,10%
93% CD105+/CD34-
CD105+/CD45-
CD34-/CD45-
Fig 9. Dental pulp stem cells phenotype after cryopreservation. Method N°1
Fig 10. Dental pulp stem cells phenotype after cryopreservation. Method N°2
5. DISCUSSION AND CONCLUSIONS Recent studies describe methods for characterization, isolation and cell culture of DPSCs (Gronthos et al/2000; Miura et al/2003; Laino et al/2005;
Iohara et al/2006; Kerkis et al/2006; Lindroos et al/2008; Pinheiro et al/2008; Suchanek et al/2009; Spath et al/2009). Few studies report DPSCs cryopreservation methods (Zhang et al/2006; Papaccio et al/2006; Perry/ et al2008; Woods et al/2009) although they do not analyze factors which may
be decisive in the effectiveness of processes, as suggested by the results of this study . There is a significant and inverse correlation between the handling time and the number of DPSC´s CD 105+. The ideal teeth are included third molars and decidous teeth. It is reported a greater differentiation capacity of mesenchymal stem cells in connective tissue of younger patients (Gronthos et al/ 2002). There is a mild and indirect relationship between the patient's age and the number of DPSC's CD105+ isolated with Miltenyi. Although the results are not conclusive due to the reduced sample size, they show important trends for an optimal protocol that must be taken in account for an effective isolation of DPSCs. 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. (Woods et al/ 2004). It is essential to evaluate the effect of two methods of cryopreservation for three different times on the viability and phenotype of mesenchymal stem cells of pulpal origin.
REFERENCES 1. Krebsbach P, Gehron R P. Dental and skeletal Stem cells: Potential Cellular Therapeutics for Craniofacial Regeneration. Journal of Dental Education. 2002, 66 (6): 766 – 773. 2. Dominici M, Le Blanc M, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8:315-317 3. Pittenger MF, et al. Multilineage potential of adult human Mesenchymal Stem cells. Science 1999; 248: 143–7. 4. Gronthos S, Mankani M, Brahim J,Robey P G, Shi S. Postnatal human dental pulp stem cells (DPSC's) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000 Dec 5; 97 (25): 13625-30. 5. Gronthos S, Brahim J, Li W. Stem cell properties of human dental pulp stem cells. J Dent Res 2002; (81):531- 35. 6. Miura M, Gronthos S, Zhao M . SHED: stem cells from human exfoliated deciduous teeth. PNAS. 2003. (100):5807–12. 7. Ulloa-Montoya F, Verfaillie C, Hu W. S . Culture Systems for Pluripotent Stem cells. Journal of Bioscience and Bioengineering. 2005. Jul; 100(1): 12- 27. 8. Kerkis I, Kerkis A, Dozortsev D, Stukart-Parsons GC, Gomes Massironi SM, Pereira LV, Caplan AI, Cerruti HF. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers Cells Tissues Organs. 2006;184 (3-4):105-16. 9. Iohara K et al. The Side Population Cells Isolated from Porcine Dental Pulp Tissue with Self-renewal and Multipotency for Dentinogénesis, Chondrogenesis, Adipogene sis and Neurogenesis. Stem Cells. 2006. 24(11). 2493-2503.
10. Pinheiro S L, Marchadier A, Donas P, Septier D, Benhamou L, Kellerman O, Goldberg M, Poliard A. An in vivo model for short term evaluation for the implantation effects of biomolecules or stem cells in the dental pulp. Open Dentistry Journal. 2008; 2: 67 -72. 11. Lindroos B, Mäenpää K, Ylikomi T, Oja H, Suuronen R, Miettinen S. Characterisation of human dental stem cells and buccal mucosa fibroblasts. Biochem Biophys Res Commun. 2008 Apr 4;368(2):329-35. 12. Suchanek J et al. Dental pulp stem cells and their characterization. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2009 Mar;153 (1):31-5. 13. Woods EJ, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. 2004. Cryobiology; 48:146-56. 14. Seo BM., Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. Recovery of Stem Cells from Cryopreserved Periodontal Ligament. J Dent Res 84 (10):907-912, 2005 15. Papaccio G, Graziano A, d'Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G. J Cell Physiol. Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. 2006 Aug; 208(2):319-25. 16. Thirumala S, Goebel WS, Woods EJ. Clinical grade adult stem cell banking. Organogenesis. 2009 Jul;5(3):143-54. 17. Zhang W, Walboomers XF, Shi S, Fan M, Jansen JA. Multilineage differentiation potential of stem cells derived from human dental pulp after cryopreservation. Tissue Eng. 2006 Oct; 12(10):2813-23. 18. Perry BC, Zhou D, Wu X, Yang FC, Byers MA, Chu TM, Hockema JJ, Woods EJ, Goebel WS. Collection, cryopreservation, and characterization of humn dental pulp-derived mesenchymal stem cells for banking and clinical use. Tissue Eng Part C Methods. 2008 Jun;14(2):149-56. 19. Woods E. J, Perry B. C, Hockema J. J, Larson L, Zhou D, Goebel W. S. Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology. 2009. 59; 150-157.