Glicose e glutamina na proliferação e viabilidade de células-tronco dentais humanas

Abstract

Introdução: Vias de crescimento, expressao genetica, sintese de macromoleculas, formacao de purinas e pirimidinas e producao de energia estao relacionadas ao metabolismo da glicose e da glutamina. Por isso, o monitoramento dos niveis destes nutrientes e comum na cultura de celulas. Uma vez que o periodo in vitro e obrigatorio na fase pre-clinica da Medicina Regenerativa, as condicoes de cultivo devem ser bem estabelecidas, sendo importante o estudo dos requerimentos nutricionais celulares. Objetivo: Investigar o efeito da glicose e da glutamina sobre a proliferacao e a viabilidade de celulas-tronco dentais humanas. Metodos: O tecido de terceiros molares foi fragmentado em explantes e cultivado ate a obtencao do numero de celulas para as etapas experimentais. As celulas foram submetidas a diferentes concentracoes de glicose e glutamina na presenca ou ausencia de soro, e em seguida, avaliadas em relacao ao numero, viabilidade pelo teste de MTT e estruturas do citoesqueleto, nucleo e mitocondrias com microscopia confocal. Resultados: Baixas concentracoes de glicose favoreceram a viabilidade e a morfologias adequada. A presenca de glutamina mostrou-se benefica apenas quando associada a glicose. A preservacao do potencial biologico em cultura e possivel na ausencia de soro, desde que os requerimentos nutricionais estejam adequados. Conclusao: O comportamento microscopico e a viabilidade celular demonstraram padroes superiores em um meio de cultura livre de soro animal e isento de glutamina. Para determinar os requerimentos de glutamina, mais investigacoes sao necessarias. As doses adequadas e a composicao de nutrientes-chave irao favorecer a selecao de uma populacao celular adequada

Introduction: Monitoring the glucose and glutamine levels is a common practice in the context of cell culture. Growth pathways, genetic expression, macromolecules synthesis, purine and pirimidine formation, and energy production are connected to glucose and glutamine metabolism. Before transplantation in Tissue Engineering and Regenerative Medicine, cells need to proliferate in vitro and the culture conditions must be well-established. In order to preserve cell biological activity and viability following transplantation, cell nutrient requirements need to be studied. Objective: To evaluate microscopic behavior, morphology and viability of human mesenchymal cells under glucose and glutamine deprivation. Materials and Methods: Human tooth tissues were minced in isolated pieces and cultured until the desired cellular proliferation for experimental phases. The cells were cultured under variations of Glucose and Glutamine in both serum presence and absence, and then those cells were evaluated according to number and viability by MTT assay. The confocal microscopy analyzed cytoskeleton, nucleus, and mitochondria integrity. Results: A low concentration of glucose favored cellular viability and microscopic behavior; the presence of glutamine in culture medium was favorable only when associated with glucose. The preservation of cell biological potential in culture is possible in serum absence provided that the nutritional requirements are adequate. Conclusion: Cell microscopic behavior and viability has demonstrated better patterns on serum-free low glucose culture medium with glutamine deprivation. Further glutamine analysis are necessary to understand its requirements to the HDSC. The employment of the correct dose and composition of key nutrients will favor an appropriate cell population selection for future transplantation.