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
Poster # 2394 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 includes applications in areas such as liver repair, spinal cord injury, 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, Munévar 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 cryopreservation, is the cellular freezing process that reduce the metabolic activity during long periods of time, preserving it viability and differentiation potential (Woods et al/2004). Its importance lies in that freezing doesn`t destroy the tissue because the technique involves cryoprotectants use that prevent the formation of ice crystals 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 researches 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
1. An in vitro study of 48 healthy dental pulp taken from subjects between 14 and 31 years of age
In vitro DPSCs Expansion:
Flow Cytometry Viability
Flow Cytometry Immunophenotyping
PHASE CONTRAST MICROSCOPY 2. The samples were mechanically and enzymatically dissociated 3. Trypan blue cell viability 4. CD 105+ cells magnetique isolation by MiniMACS Miltenyi 5. In vitro CD 105+ expansion
DPSCs morphology in vitro. 40x
Wharton’s jelly MSC in vitro 10x
Cell viability percentage of hDPSCs by Papaccio et al/2006 and Kamath et al/2007 cryopreservation methods.
6. Two methods of DPSCs cryopreservation; Papaccio et al/2006 and Kamath et al/2007; 3 different times: 1,7 y 30 days
hDPSCs phenotype percentage by Papaccio et al/2006 and Kamath et al/2007 cryopreservation methods.
5. DISCUSSION AND CONCLUSIONS Human dental pulp morphology of fibroblast in vitro 10x
DPSCs colonies forming unit (CFU) 10x
Post-cryopreservation cell viability evaluation by flow cytometry
♦ 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 postcryopreservation 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.
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♦The expression of CD73 marker was higher when the Papaccio et al/2006 method was used within 1 and 7 days post-cryopreservation.
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7. Post-cryopreservation cell viability evaluation by flow cytometry with 7AAD– .
♦ 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.
8. Post-cryopreservation cell phenotype evaluation CD105+,CD73+,CD45-, CD34-
♦ It is essential to evaluate the viability and to determine the phenotype of hDPSCs during longer cryopreservation times.
3. MATERIALS AND METHODS 9. Statistical analysis: Student T test , U Mann Whitney P≤0.05 Control-: Fibroblast Control+: Wharton`s jelly H.U.C
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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 Viability at 7 days post-cryopreservation method Papaccio 2006. Figure D: DPSCs Viability at 7 days post-cryopreservation method Kamath2007 .
DPSCs cell viability comparison by two methods of cryopreservation
♦ 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.)
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Post-cryopreservation cell phenotype evaluation
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Figure A, B: DPSCs phenotype CD105+/CD34-/CD45 at 24 hours, 7 days Postcryopreservation method Papaccio 2006. Figure C, D: DPSCs phenotype CD105+/CD34-/CD45– at 24 hours, 7 days Post cryopreservation method Kamath 2007
This study was supported by an El Bosque University research grant PCI 120/2010
CD105+/CD73+ DPSCs Phenotype comparison by two methods of cryopreservation
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