Revista de Odontologia da UNESP
https://revodontolunesp.com.br/article/doi/10.1590/S1807-25772012000600008
Revista de Odontologia da UNESP
Original Article

Yield of human periodontal ligament mesenchymal cells under different protocols of cryopreservation

Rendimento de células mesenquimais do ligamento periodontal humano submetidas a diferentes protocolos de criopreservação

Soares, Diego Moura; Ginani, Fernanda; Barboza, Carlos Augusto Galvão

Downloads: 0
Views: 987

Abstract

Cryopreservation aims to stop reversibly the biological functions of living tissues at low temperatures, and is an important resource for the storage of human cells for later use. Aim: To assess the proliferation of mesenchymal cells from human periodontal ligament cryopreserved by two different protocols. Method: Periodontal ligament cells were obtained from third molars with an indication for surgical removal. After processing, cells were grown and maintained at 37 °C in 5% CO2 until they reached 70-90% confluency, with medium changing every three days. In the first passage cells were divided into two groups, according to the protocol used: Group –80 °C - cryopreserved in ultrafreezer for 45 days, Group –196 °C - cryopreserved in liquid nitrogen for 45 days. After this time, cells from both groups were thawed and plated for the experiment. The growth curve of the groups was drawn from counting cells in a Neubauer chamber and by the MTT assay method, in the intervals of 24, 48 and 72 hours. The data were analyzed using the Mann-Whitney test with a significance level of 5%. Result: There was an upward cell growth in both protocols used, but a higher proliferative rate was observed in group cryopreserved in liquid nitrogen (p < 0.05). Conclusion: Cryopreservation has proven to be an effective technique for the storage and of mesenchymal cells from the periodontal ligament, especially when stored at a temperature of –196 °C.

Keywords

Periodontal ligament, cryopreservation, cell proliferation.

Resumo

A técnica de criopreservação tem como característica cessar reversivelmente todas as funções biológicas dos tecidos vivos em baixas temperaturas e tem sido aplicada a diversas células humanas, visando à sua utilização posterior. Objetivo: Avaliar a proliferação de células mesenquimais do ligamento periodontal humano após a criopreservação por dois diferentes protocolos. Método: As células do ligamento periodontal foram obtidas a partir de dois dentes (terceiros molares) hígidos, com indicação de remoção cirúrgica. Após o processamento, as células foram cultivadas em placas de Petri e mantidas a 37 °C em 5% de CO2, até atingirem 70-90% de confluência, com troca de meio a cada três dias. Na primeira passagem, as células foram divididas em dois grupos e criopreservadas: Grupo –80 °C – criopreservação em ultrafreezer por 45 dias; Grupo –196 °C – criopreservação em nitrogênio líquido por 45 dias. Decorrido esse tempo, as células dos dois grupos foram descongeladas e plaqueadas para o experimento. A curva de crescimento dos grupos estudados foi traçada a partir de contagem em Câmara de Neubauer e pelo método de ensaio do MTT, nos intervalos de 24, 48 e 72 horas. Os resultados foram analisados por meio do teste de Mann‑Whitney, com nível de significância de 5%. Resultado: Verificou-se um crescimento ascendente nos dois protocolos utilizados, porém uma maior taxa proliferativa foi verificada no grupo criopreservado em nitrogênio líquido (p < 0,05). Conclusão: Ambos os protocolos de criopreservação estudados foram eficazes, porém a criopreservação em nitrogênio líquido (–196 °C) manteve uma maior taxa de proliferação celular em todos os intervalos de tempo.

Palavras-chave

Ligamento periodontal, criopreservação, proliferação de células.

References



1. Barry FP. Biology and clinical applications of mesenchymal stem cells. Birth Defects Res C Embryo Today. 2003;69:250-6. PMid:14671778. http://dx.doi.org/10.1002/bdrc.10021

2. Hoffman LM, Carpenter MK. Characterization and culture of human embryonic stem cells. Nat Biotechnol. 2005;23:699-708. PMid:15940242. http://dx.doi.org/10.1038/nbt1102

3.Freshney IR, Stacey GN, Aurebach JM. Culture of human stem cells: culture of specialized cells. New York: Wiley; 2007. PMid:17987034 PMCid:2360285. http://dx.doi.org/10.1002/9780470167526

4. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97:13625-30. PMid:11087820 PMCid:17626. http://dx.doi.org/10.1073/pnas.240309797

5. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA. 2003;100:5807-12. PMid:12716973 PMCid:156282. http://dx.doi.org/10.1073/pnas.0937635100

6. Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, et al. Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP). Arch Oral Biol. 2011;56:709-21. PMid:21227403. http://dx.doi.org/10.1016/j.archoralbio.2010.12.008

7. Kramer PR, Nares S, Kramer SF, Grogan D, Kaiser M. Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro. J Dent Res. 2004;83:27-34. PMid:14691109. http://dx.doi.org/10.1177/154405910408300106

8. Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. Recovery of stem cells from cryopreserved periodontal ligament. J Dent Res. 2005;84:907-12. PMid:16183789. http://dx.doi.org/10.1177/154405910508401007

9. Chen SC, Marino V, Gronthos S, Bartold PM. Location of putative stem cells in human periodontal ligament. J Periodontal Res. 2006;41:547‑53. PMid:17076780. http://dx.doi.org/10.1111/j.1600-0765.2006.00904.x

10. De Santis GC, Lima Prata K. Criopreservação de células-progenitoras hematopoéticas. Medicina (Ribeirão Preto). 2009;42:36-47.

11. Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997;64:278-94. http://dx.doi.org/10.1002/(SICI)1097-4644(199702)64:2<278::AID-JCB11>3.0.CO;2-F

12. Gonda K, Shigeura T, Sato T, Matsumoto D, Suga H, Inoue K, et al. Preserved proliferative capacity and multipotency of human adipose-derived stem cells after long-term cryopreservation. Plast Reconstr Surg. 2008;121:401-10. PMid:18300956. http://dx.doi.org/10.1097/01.prs.0000298322.70032.bc

13. Lima Prata K, de Santis GC, Orellana MD, Palma PV, Brassesco MS, Covas DT. Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions. Cytotherapy. 2012;14:694-700. PMid:22519634.

14. Papaccio G, Graziano A, d'Aquino R, Graziano MF, Pirozzi G, Menditti D, et al. Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol. 2006;208:319-25. PMid:16622855. http://dx.doi.org/10.1002/jcp.20667

15. Vasconcelos RG, Ribeiro RA, Vasconcelos MG, Lima KC, Barboza CA. In vitro comparative analysis of cryopreservation of undifferentiated mesenchymal cells derived from human periodontal ligament. Cell Tissue Bank. 2011. http://dx.doi.org/10.1007/s10561-011-9271-3

16. Mazur P. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J Gen Physiol. 1963;47:347‑69. PMid:14085017 PMCid:2195343. http://dx.doi.org/10.1085/jgp.47.2.347

17. Temmerman L, Dermaut LR, De Mil M, Van Maele G, Beele H, De Pauw GA. Influence of cryopreservation on human periodontal ligament cells in vitro. Cell Tissue Bank. 2008;9:11-8. PMid:17541731. http://dx.doi.org/10.1007/s10561-007-9047-y

18. Oh YH, Che ZM, Hong JC, Lee EJ, Lee SJ, Kim J. Cryopreservation of human teeth for future organization of a tooth bank--a preliminary study. Cryobiology. 2005;51:322-9. PMid:16297377. http://dx.doi.org/10.1016/j.cryobiol.2005.08.008

19. Ding G, Wang W, Liu Y, An Y, Zhang C, Shi S, et al. Effect of cryopreservation on biological and immunological properties of stem cells from apical papilla. J Cell Physiol. 2010;223:415-22. PMid:20082304.

20. Woods EJ, Perry BC, Hockema JJ, Larson L, Zhou D, Goebel WS. Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology. 2009;59:150-7. PMid:19538953 PMCid:2757034. http://dx.doi.org/10.1016/j.cryobiol.2009.06.005

21. Lee SY, Chiang PC, Tsai YH, Tsai SY, Jeng JH, Kawata T, et al. Effects of cryopreservation of intact teeth on the isolated dental pulp stem cells. J Endod. 2010;36:1336-40. PMid:20647092. http://dx.doi.org/10.1016/j.joen.2010.04.015

22. Thirumala S, Gimble JM, Devireddy RV. Transport phenomena during freezing of adipose tissue derived adult stem cells. Biotechnol Bioeng. 2005;92:372-83. PMid:16155954. http://dx.doi.org/10.1002/bit.20615

23. Thirumala S, Zvonic S, Floyd E, Gimble JM, Devireddy RV. Effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells. Biotechnol Prog. 2005;21:1511-24. PMid:16209556. http://dx.doi.org/10.1021/bp050007q

24. Hubel A. Parameters of cell freezing: Implications for the cryopreservation of stem cells. Transfus Med Rev. 1997;11:224-33. PMid:9243775. http://dx.doi.org/10.1053/tmrv.1997.0110224

588019337f8c9d0a098b5022 rou Articles
Links & Downloads

Rev. odontol. UNESP

Share this page
Page Sections