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MÉTODO DE CRIOPRESERVAÇÃO DE UMA MONOCAMADA DE QUERATINÓCITOS COM BASE EM SISTEMAS EUTÉTICOS PROFUNDOS NATURAIS
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O principal objetivo deste projeto foi explorar o potencial dos Sistemas Eutéticos Profundos Naturais (NADES) como agentes crioprotetores (CPAs) alternativos na criopreservação de estruturas celulares complexas, como o tecido cutâneo. Este estudo baseia-se em resultados prévios que demonstraram a eficácia dos NADES na criopreservação de células em suspensão, e visa testar a sua aplicação na criopreservação de monocamadas de queratinócitos humanos (células HaCaT). Para isso, foram otimizados protocolos de formação de monocamadas celulares e avaliada a capacidade protetora de diferentes sistemas durante o congelamento e descongelamento, comparando-os com o dimetilsulfóxido (DMSO), o CPA padrão, conhecido pela sua eficácia, mas cuja toxicidade é um fator limitante.
As soluções crioprotetoras à base de NADES foram caraterizadas por calorimetria diferencial de varrimento (DSC) e microscopia ótica com luz polarizada (POM). Em termos de citotoxicidade, as soluções não se mostraram tóxicas durante o período de congelamento/descongelamento. Foram realizados diversos ensaios de criopreservação das monocamadas celulares em placas de cultura para avaliar a capacidade protetora das soluções com os NADES.
Os resultados indicaram que, embora os NADES apresentem propriedades promissoras como CPAs, ainda enfrentam desafios significativos na preservação de monocamadas celulares, principalmente devido à formação de cristais durante o protocolo de congelamento, observados por POM, e à pouca eficácia do protocolo de descongelamento utilizado, relacionada com os tempos prolongados de descongelamento. A análise térmica demonstrou que os NADES influenciam as propriedades térmicas do meio de cultura, reduzindo as temperaturas
de fusão (Tm) e de cristalização (Tc). Contudo, em todos os ensaios de criopreservação da monocamada, a viabilidade celular permaneceu baixa (entre 20% e 30%), mesmo com diversas otimizações feitas ao protocolo.
Resumindo, os resultados mostraram que, apesar do potencial dos NADES como CPAs, são necessárias mais otimizações neste protocolo de criopreservação para superar desafios como a formação de cristais de gelo e os tempos prolongados de descongelamento, que levam a fenómenos, como a recristalização e consequentemente a dano celular. No entanto, este trabalho contribui como ponto de partida para futuras inovações na criopreservação bem-sucedida de tecidos, em particular do tecido cutâneo, altamente procurado na medicina regenerativa.
The main objective of this project was to explore the potential of Natural Deep Eutectic Systems (NADES) as alternative cryoprotectant agents (CPAs) in the cryopreservation of complex cellular structures, such as skin tissue. This study is based on previous results demonstrating the efficacy of NADES in the cryopreservation of cells in suspension and aimed to test their application in the cryopreservation of human keratinocyte monolayers (HaCaT cells). To this end, protocols for establishing cell monolayers were optimized. The protective capacity of different systems during freezing and thawing was evaluated, comparing them with dime- thyl sulfoxide (DMSO), the standard CPA, which is known for its efficacy but whose toxicity is a limiting factor. The NADES-based cryoprotectant solutions were characterized by differential scan- ning calorimetry (DSC) and polarized light microscopy (POM). In terms of cytotoxicity, the solutions were not toxic during the freeze-thaw period. Several cryopreservation assays were carried out on the cell monolayers in culture plates to assess the protective capacity of the solutions containing the NADES. The results indicated that, although NADES showed promising properties as CPAs, they still face significant challenges in preserving cell monolayers. This occurs mainly due to crystal formation during the freezing protocol, observed in the POM analyses, and the poor effectiveness of the thawing protocol used, related to prolonged thawing times. Thermal anal- ysis showed that NADES influence the thermal properties of the culture medium, reducing the melting (Tm) and crystallization (Tc) temperatures. However, in all the monolayer cryo- preservation assays, the cell viability remained low (between 20% and 30%), even after differ- ent optimization steps made to the protocol. In summary, the results showed that, despite the potential of NADES as CPAs, further optimization is needed in the cryopreservation protocol designed in this project, to overcome the challenges such as the formation of ice crystals and prolonged thawing times which leads to recrystallization and consequently cell damage. Nevertheless, this work contributed as a starting point for future innovations in tissue cryopreservation, particularly skin tissue, which is a key area of interest within regenerative medicine.
The main objective of this project was to explore the potential of Natural Deep Eutectic Systems (NADES) as alternative cryoprotectant agents (CPAs) in the cryopreservation of complex cellular structures, such as skin tissue. This study is based on previous results demonstrating the efficacy of NADES in the cryopreservation of cells in suspension and aimed to test their application in the cryopreservation of human keratinocyte monolayers (HaCaT cells). To this end, protocols for establishing cell monolayers were optimized. The protective capacity of different systems during freezing and thawing was evaluated, comparing them with dime- thyl sulfoxide (DMSO), the standard CPA, which is known for its efficacy but whose toxicity is a limiting factor. The NADES-based cryoprotectant solutions were characterized by differential scan- ning calorimetry (DSC) and polarized light microscopy (POM). In terms of cytotoxicity, the solutions were not toxic during the freeze-thaw period. Several cryopreservation assays were carried out on the cell monolayers in culture plates to assess the protective capacity of the solutions containing the NADES. The results indicated that, although NADES showed promising properties as CPAs, they still face significant challenges in preserving cell monolayers. This occurs mainly due to crystal formation during the freezing protocol, observed in the POM analyses, and the poor effectiveness of the thawing protocol used, related to prolonged thawing times. Thermal anal- ysis showed that NADES influence the thermal properties of the culture medium, reducing the melting (Tm) and crystallization (Tc) temperatures. However, in all the monolayer cryo- preservation assays, the cell viability remained low (between 20% and 30%), even after differ- ent optimization steps made to the protocol. In summary, the results showed that, despite the potential of NADES as CPAs, further optimization is needed in the cryopreservation protocol designed in this project, to overcome the challenges such as the formation of ice crystals and prolonged thawing times which leads to recrystallization and consequently cell damage. Nevertheless, this work contributed as a starting point for future innovations in tissue cryopreservation, particularly skin tissue, which is a key area of interest within regenerative medicine.
Descrição
Palavras-chave
Criopreservação tecido cutâneo agentes crioprotetores NADES DMSO