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

Poster #566

1. INTRODUCTION

Due to the high prevalence of dental and periodontal pathologies that cause irreversible damage to teeth and maxillary structures, it is nec-essary to approach new therapeutical strategies. The human dental pulp Stem cells (DPSCs), basic in the mechanisms of tissue develop-ment and regeneration (Gronthos et al/ 2000 Shi et al/ 2001) are fun-damental 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, par-ticularly those related to the conservation of these ex-vivo cells at ex-tremely low temperatures.

Pool Sample

Patient age *

Tooth Obtention time of the samples **

Processing time of the samples **

premolar 3rd Molar

Erupted Included

1 28 2 4 5

2 24 1 2 2 5

3 28 2 3 5

4 27 2 2 5

5 29 2 1 5

6 29 2 2 5

7 31 2 12 4

8 30 2 14 4

9 18 4 15 4

10 26 2 2 4

Total ¥ and/or average∞

27 +3.74∞ 12¥ 9¥ 2¥ 5.7 +5.9∞ 4.6+0.71∞

* years of age ** hours

Table 1. Detailed description of the determining factors evaluated.

4.RESULTS

The analysis demonstrate an inverse, moderate and statistically significant association between the collection and the process-ing 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.

1A 1B

Fig 1. Fibroblast-like morphology of DPSCs confluent after 18 days of observation in primary culture.

Phase contrast microscope. (1A): 40x (1B): 20X

POOL

sample

N° of pulp cells/ml

isolated with

MEDIMACHINE

N° of stem cells/ ml

Isolated with

MILTENYI

% CD 105

+

N° of wells for

POOL sample

1 280.000 45.000 16 9

2 410.000 90.000 21 18

3 255.000 40.000 15 8

4 250.000 30.000 12 6

5 425.000 50.000 12 10

6 225.000 25.000 11 5

7 260.000 52.000 20 11

8 220.000 30.000 14 6

9 115.000 20.000 17 4

10 240.000 40.000 17 8

Average

– DS 268.000+90.590 cels/ml 35.285+12.338 cels/ml 16%+0.03 8.5+4

Table 2. Detailed description of expected results

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 obtai-ned after mechanical disgregation with Medimachine. (B): Correlation between processing time of the sam-ples (h) and the number of pulp cells obtained after mechanical disgregation with Medimachine.

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.

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2. OBJECTIVE

To establish the isolation method for future cryopreservation of DPSCs determining factors as age, tooth type, collection and process-ing times that can influence their quality and quantity.

Fig 2. (2A): Positive control mesenchymal stem cells from Whaton’s Jelly of Umbilical Cord. (2B): Negative control human fibroblasts (40X)

2B

DPSCs VIABILITY & PHENOTYPE AFTER CRYOPRESERVATION

Fig 6. DPSCs viability and phenotype CD105+/CD34-/CD45– after 24 hours in cryopreservation. Method N° 2 Fig 5. DPSCs viability and phenotype CD105+/CD34-/CD45– after 24 hours in cryopreservation. Method N°1

Fig 4. Method N°1. Negative control without antibodies

2A

Fig 7. DPSCs Viability and phenotype CD105+/CD34-/CD45– after 7 days in cryopreservation. Method N°1 Fig 8. DPSCs viability and phenotype CD105+/CD34-/CD45– after 7 days in cryopreservation. Method N°2

3. MATERIALS AND METHODS

DPSCs Phenotype

Method 1 1 day 7 days

CD105+/CD34- 99% 100%

CD105+/CD45- 95,40% 98,10%

CD34-/CD45- 96,60% 98,80% 93%

94%

95%

96%

97%

98%

99%

100%

CD105+/CD34- CD105+/CD45- CD34-/CD45-

DPSCs phenotype 1 day

DPSCs phenotype 7 days

DPSCs Phenotype

Method 2 1 day 7 days

CD105+/CD34- 95% 98%

CD105+/CD45- 93,00% 96,30%

CD34-/CD45- 99,30% 99,10%

Fig 9. Dental pulp stem cells phenotype after cryopreservation. Method N°1 Fig 10. Dental pulp stem cells phenotype after cryopreservation. Method N°2

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4. CD105+ cell cultureCD105+ cell culture