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 (Krebsbach P, Gehron R P/2002) 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. Sin embargo, hay varios desafíos en relación con la calidad y seguridad en las aplicaciones clínicas de las células stem adultas, en particular aquellos relacionados con la conservación de estas células ex-vivo a temperaturas extremadamente bajas (Thirumala et al/2009)
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.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
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 **
Processing time of the samples **
4 2 3 2 1 2 12 14 15 2
5 5 5 5 5 5 4 4 4 4
5.7 +5.9∞
4.6+0.71∞
* years of age ** hours
POOL sample
1B
Fig 1. Fibroblast-like morphology of DPSCs confluent after 18 days of observation in primary culture. Phase contrast microscope. (1A): 40x (1B): 20X
2B
2A
Fig 2. (2A): Positive control mesenchymal stem cells from Whaton’s Jelly of Umbilical Cord. (10X) (2B): Negative control human fibroblasts (40X)
Table 1. Detailed description of the determining factors evaluated.
N° of pulp cells/ml isolated with MEDIMACHINE
1A
N° of stem cells/ ml Isolated with MILTENYI
% CD 105 +
1 280.000 45.000 16 2 410.000 90.000 21 3 255.000 40.000 15 4 250.000 30.000 12 5 425.000 50.000 12 6 225.000 25.000 11 7 260.000 52.000 20 8 220.000 30.000 14 9 115.000 20.000 17 10 240.000 40.000 17 Average 35.285+12.338 cels/ 268.000+90.590 cels/ml 16%+0.03 – DS ml
N° of wells for POOL sample 9 18 8 6 10 5 11 6 4 8 8.5+4
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.
Table 2. Detailed description of expected results (DESCRIBE LOS RESULTADOS? A que te refieres?)
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 cryopreserved with method N. 1.
Fig 7. DPSCs Viability and phenotype CD105+/CD34-/CD45– 7 days cryopreserved with method N.1.
Fig 6. DPSCs viability and phenotype CD105+/CD34-/CD45– after 24 hours cryopreserved with method N. 2.
Fig 8. DPSCs viability and phenotype CD105+/CD34-/CD45– after 7 days cryopreserved with method N.2
5. DISCUSSION AND CONCLUSIONS Estudios recientes describen métodos de caracterización, aislamiento y cultivo de DPSCs (Gronthos/2000; Miura/2003; Laino/2005; Iohara/2006; Kerkis/2006; Lindroos/2008; Pinheiro/2008; Suchanek/2009; Spath/2009). Pocos estudios reportan métodos de criopreservación de DPSCs (Seo et al/2005; Papaccio/2006; Perry/2008; Woods/2009) sin embargo no analizan factores que pueden ser determinantes en la efectividad de los procesos, como sugieren los resultados de este estudio. There is a significant and inverse correlation between the handling time and the number of DPSC´s CD 105+ isolated. 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/ 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. Munévar J C , Forero J, Lafaurie G. In vitro phenotype of mesenchymal stem cells in human dental pulp. International Association of Dental Research. 2009. Miami. USA. 10. Iohara K et al. The Side Population Cells Isolated from Porcine Dental Pulp Tissue with Self-renewal and Multipotency for Dentinogénesis, Chondrogenesis, Adipogenesis and Neurogenesis. Stem Cells. 2006. 24(11). 2493-2503. 11. 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. 12. 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. 13. 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. 14. Woods EJ, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. 2004. Cryobiology; 48:146-56. 15. 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 16. 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. 17. Thirumala S, Goebel WS, Woods EJ. Clinical grade adult stem cell banking. Organogenesis. 2009 Jul;5(3):143-54. 18. Perry BC, Zhou D, Wu X, Yang FC, Byers MA, Chu TM, Hockema JJ, Woods EJ, Goebel WS. Collection, cryopreservation, and characterization of human 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.