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
http://dx.doi.org/10.1590/S1807-25772012000600008
Rev. odontol. UNESP, vol.41, n6, p.415-419, 2012
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.
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