I1. NTRODUCTION

Mesenchymal stem cells (MSC) are multipotent and can repair many different tissues, considered as key players in the future treatment of diseases such as Parkinson’s Disease, Alzheimer’s Disease, and Type I Diabetes. Other research with MSC cells in-cludes applications in areas such as liver repair, spinal cord inju-ry, MS, and stroke rehabilitation. These kind of cells are located in bone marrow, umbilical cord, adipose tissue but also can be found in human dental pulp (Gronthos et al/2002). Pulp stem cells have an elevated proliferative, self-renewal and multilineage diferentiation potential into osteoblasts, fibroblasts, cementoblasts and odontoblasts that have been isolated throught the CD105+, CD73+, STRO1, CD146+, CD34- and CD45- expression (Prockop D J, 1997, Pittenger et al/1999, Barry F P et al/2004, Mu-névar J C y col, 2005). There is a growing interest in dental pulp stem cells (DPSCs) cryobiology due to its therapeutic potential in the clinical practice. (Krebsbach et al/2002). DPSCs cryopreserva-tion, is the cellular freezing process that reduce the metabolic ac-tivity during long periods of time, preserving it viability and differ-entiation potential (Woods et al/2004). Its importance lies in that freezing doesn`t destroy the tissue because the technique in-volves cryoprotectants use that prevent the formation of ice crys-tals that pierce the cell. 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. There are several re-searches concerning DPSCs cryopreservation; however, until our knowledge no one compares two cryopreservation methods. 2. OBJECTIVE

To evaluate the effect of two cryopreservation methods in three different times on the DPSCs viability and phenotype

3. MATERIALS AND METHODS

Poster # 2394

hDPSCs phenotype percentage by Papaccio et al/2006

and Kamath et al/2007 cryopreservation methods.

HUMAN DENTAL PULP MESENCHYMAL STEM CELLS CRYOPRESERVATION N.T JIMÉNEZ-ORTEGON, J.C MUNEVAR-NINO, N GUTIERREZ, M.C TAMAYO-MUNOZ

Unidad de Investigación Básica Oral U.I.B.O. Universidad el Bosque. Bogotá. Colombia .

munervarjuan@unbosque.edu.co

Wharton’s jelly MSC in vitro 10x DPSCs morphology in vitro. 40x

DPSCs colonies forming unit (CFU) 10x Human dental pulp morphology of fibroblast

in vitro 10x

PHASE CONTRAST MICROSCOPY

Post-cryopreservation cell viability evaluation by flow cytometry

Figure A: DPSCs Viability at 24 hours post-cryopreservation method Papaccio 2006 , Figure B:

DPSCs Viability at 24 hours post-cryopreservation method Kamath2007. Figure C: DPSCs Via-

bility at 7 days post-cryopreservation method Papaccio 2006. Figure D: DPSCs Viability at 7

days post-cryopreservation method Kamath2007 .

Figure A, B: DPSCs phenotype CD105+/CD34-/CD45 at 24 hours, 7 days Post-

cryopreservation method Papaccio 2006.

Figure C, D: DPSCs phenotype CD105+/CD34-/CD45– at 24 hours, 7 days Post

cryopreservation method Kamath 2007

A B

C D

DPSCs cell viability comparison by two methods of cryopreservation

A B

C D

CD105+/CD73+ DPSCs Phenotype comparison by two methods of cryopreservation

Post-cryopreservation cell phenotype evaluation

Cell viability percentage of hDPSCs by Papaccio et al/2006

and Kamath et al/2007 cryopreservation methods.

In vitro DPSCs Expansion:

This study was supported by an El Bosque University research grant PCI 120/2010

Flow Cytometry Immunophenotyping

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5. DISCUSSION AND CONCLUSIONS

♦ The Papaccio et al/2006 method showed the higher cell viability after 1 month post-cryopreservation when it

was compared to Kamath et al/2007 method, however, the Kamath et al/2007 method showed better results of

cell viability after 1 and 7 days post-cryopreservation (65.5% and 56% respectively).

♦ There were a higher percentage of CD105+/CD34– hDPSCs expression-markers after 1 and 7 days post-

cryopreservation by the Papaccio et al/2006 method.

♦ The Papaccio et al/2006 method showed a higher expression of CD105+/CD45– markers at the 3 different

cryopreservation times than the Kamath et al/2007 method.

♦The expression of CD73 marker was higher when the Papaccio et al/2006 method was used within 1 and 7

days post-cryopreservation.

♦ Higher amounts of hDPSCs cryopreserved cells when the Kamath et al/2007 method was used showed the

higher expression of CD105+/CD34–; CD105+/CD45– , CD73+ after 1 month post-cryopreservation.

♦ It is essential to evaluate the viability and to determine the phenotype of hDPSCs during longer cryopreser-

vation times.

♦ We recommend in further studies to use hDPSCs markers such as STRO-1, CD 146, CD105, CD73, CD34

and CD45 which had recommended by the international stem cells societies (International Stem Cell Research

Society, International Society for Cell Therapy.)

Flow Cytometry Viability

4 RESULTS

1. An in vitro study of 48 healthy dental pulp taken from subjects be-

tween 14 and 31 years of age

4. CD 105+ cells magnetique isolation by MiniMACS Miltenyi

6. Two methods of DPSCs cryopreservation; Papaccio et al/2006 and

Kamath et al/2007; 3 different times: 1,7 y 30 days

2. The samples were mechanically and enzymatically dissociated

7. Post-cryopreservation cell viability evaluation by flow cytometry

with 7AAD– .

8. Post-cryopreservation cell phenotype evaluation

CD105+,CD73+,CD45-, CD34-

3. Trypan blue cell viability

5. In vitro CD 105+ expansion

9. Statistical analysis: Student T test , U Mann Whitney P≤0.05

Control-: Fibroblast Control+: Wharton`s jelly H.U.C