Efeitos das diferentes soluções de congelação sobre a criopreservação de células-tronco mesenquimais caninas
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Data
2022-07-27
Autores
Santos, Bruna Aparecida dos
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Universidade Estadual Paulista (Unesp)
Resumo
Para utilizar as células-tronco mesenquimais caninas (CTMs) em terapias é imprescindível que sejam formados criobancos de armazenamento celular, sendo assim, a metodologia da criopreservação de CTMs necessita de estudos, uma vez que o efeito da congelação e dos agentes crioprotetores sobre as células obtidas de tecido adiposo canino ainda é pouco conhecido. Desta forma, o objetivo deste estudo foi avaliar os efeitos de diferentes tipos de soluções na criopreservação das CTMs, sobre a estrutura, viabilidade, proliferação celular, ultraestrutura e expressão gênica, bem como no seu desenvolvimento pós-descongelação. Para isto, foram isoladas células-tronco de 12 cadelas. As amostras foram divididas em 5 tratamentos: não criopreservado (controle) = NCRIO; 90% SFB (Soro fetal Bovino) com 10% DMSO = SFB; Meio de cultivo DMEM/F12 alta glicose acrescido com 20% SFB e 10% DMSO = DMEM/F12; DMEM/F12 alta glicose, acrescido com 5%PVA, 5% de ITS, 5% de BSA e 10% DMSO = PVA/ITS/BSA e BambankerTM = BAM. Sendo que em todos foram utilizados o Dimetilsufóxido (DMSO) como agente crioprotetor principal. As células foram avaliadas antes da criopreservação, pós-descongelação imediata e após o recultivo. Foi realizada uma análise de custo das diferentes soluções. A viabilidade e apoptose celular foram avaliadas pelo teste de exclusão com o corante Trypan blue e anexina V; a proliferação celular, pela curva de crescimento; a ultraestrutura em microscópio eletrônico de transmissão, a expressão gênica por RT-qPCR e a análise de custo através de pesquisa de mercado. Todos os dados foram submetidos a análise estatística pelo software SAS versão 9.4. Os resultados mostraram que as CTMs criopreservadas com o meio comercial foi superior aos demais quanto ao número de células e a proliferação celular, mas foi similar aos grupos NCRIO e PVA/ITS/BSA quanto a viabilidade celular. Embora não tenham sido observadas alterações morfológicas significativas entre os tratamentos de criopreservação, a ultraestrutura celular variou de acordo com os componentes do meio, no qual a morfologia mais próxima da encontrada nas células frescas foi observada nos grupos PVA/ITS/BSA e BAM. A expressão gênica demonstrou que as células apresentaram diferenças quanto aos tratamentos para os transcritos CD44, TGFB1, CXCL12 e IDO. Neste estudo foi observado que embora o tratamento com a marca comercial tenha sido superior aos demais crioprotetores, as outras combinações de substâncias crioprotetoras testadas foram eficientes na criopreservação das CTMs caninas, sendo alternativas que podem ser utilizadas. Todos os tratamentos possibilitaram a retomada das funções celulares normais após a descongelação e recultivo. E as soluções autoformuladas são economicamente mais viáveis que a marca comercial. Esses resultados são importantes para o estabelecimento de protocolos de criopreservação de CTMs nessa espécie, pois aumentam as possibilidades de escolha da solução crioprotetora e também permite o uso de soluções com custos mais acessíveis e sem componentes contaminantes como o SFB.
In order to use canine mesenchymal stem cells (MSCs) in therapies, it is essential that cryobanks of cell storage are formed, therefore, the methodology of cryopreservation of MSCs needs studies, since the effect of freezing and cryoprotective agents on the cells obtained from canine adipose tissue is still poorly understood. Thus, the aim of this study was to evaluate the effects of different types of solutions in the cryopreservation of MSCs, on the structure, viability, cell proliferation, ultrastructure and gene expression, as well as on their post-thawing development. For this, stem cells from 12 female dog were isolated. The samples were divided into 5 treatments: not cryopreserved (control) = NCRIO; 90% FBS (Fetal Bovine Serum) with 10% DMSO = FBS; High glucose DMEM/F12 culture medium plus 20% FBS and 10% DMSO = DMEM/F12; DMEM/F12 high glucose plus 5%PVA, 5% ITS, 5% BSA and 10% DMSO = PVA/ITS/BSA and BambankerTM = BAM. In all, Dimethylsuffoxide (DMSO) was used as the main cryoprotective agent. Cells were evaluated before cryopreservation, immediate post-thawing and after reculture. A cost analysis of the different solutions was carried out. Cell viability and apoptosis were evaluated by the Trypan blue dye and annexin V exclusion test; cell proliferation, by the growth curve; ultrastructure in transmission electron microscope, gene expression by RT-qPCR and cost analysis through market research. All data were submitted to statistical analysis using the SAS software version 9.4. The results showed that the MSCs cryopreserved with the commercial medium was superior to the others in terms of cell number and cell proliferation, but was similar to the NCRIO and PVA/ITS/BSA groups in terms of cell viability. Although no significant morphological changes were observed between the cryopreservation treatments, the cellular ultrastructure varied according to the components of the medium, in which the morphology closest to that found in fresh cells was observed in the PVA/ITS/BSA and BAM groups. The gene expression showed that the cells showed differences regarding the treatments for the CD44, TGFB1, CXCL12 and IDO transcripts. In this study, it was observed that although the treatment with the commercial brand was superior to other cryoprotectants, the other combinations of cryoprotective substances tested were efficient in the cryopreservation of canine MSCs, being alternatives that can be used. All treatments allowed the resumption of normal cell functions after thawing and reculture. And self-formulated solutions are more economically viable than branded. These results are important for the establishment of cryopreservation protocols for MSCs in this species, as they increase the possibilities of choosing the cryoprotectant solution and also allow the use of more affordable solutions without contaminating components such as FBS.
In order to use canine mesenchymal stem cells (MSCs) in therapies, it is essential that cryobanks of cell storage are formed, therefore, the methodology of cryopreservation of MSCs needs studies, since the effect of freezing and cryoprotective agents on the cells obtained from canine adipose tissue is still poorly understood. Thus, the aim of this study was to evaluate the effects of different types of solutions in the cryopreservation of MSCs, on the structure, viability, cell proliferation, ultrastructure and gene expression, as well as on their post-thawing development. For this, stem cells from 12 female dog were isolated. The samples were divided into 5 treatments: not cryopreserved (control) = NCRIO; 90% FBS (Fetal Bovine Serum) with 10% DMSO = FBS; High glucose DMEM/F12 culture medium plus 20% FBS and 10% DMSO = DMEM/F12; DMEM/F12 high glucose plus 5%PVA, 5% ITS, 5% BSA and 10% DMSO = PVA/ITS/BSA and BambankerTM = BAM. In all, Dimethylsuffoxide (DMSO) was used as the main cryoprotective agent. Cells were evaluated before cryopreservation, immediate post-thawing and after reculture. A cost analysis of the different solutions was carried out. Cell viability and apoptosis were evaluated by the Trypan blue dye and annexin V exclusion test; cell proliferation, by the growth curve; ultrastructure in transmission electron microscope, gene expression by RT-qPCR and cost analysis through market research. All data were submitted to statistical analysis using the SAS software version 9.4. The results showed that the MSCs cryopreserved with the commercial medium was superior to the others in terms of cell number and cell proliferation, but was similar to the NCRIO and PVA/ITS/BSA groups in terms of cell viability. Although no significant morphological changes were observed between the cryopreservation treatments, the cellular ultrastructure varied according to the components of the medium, in which the morphology closest to that found in fresh cells was observed in the PVA/ITS/BSA and BAM groups. The gene expression showed that the cells showed differences regarding the treatments for the CD44, TGFB1, CXCL12 and IDO transcripts. In this study, it was observed that although the treatment with the commercial brand was superior to other cryoprotectants, the other combinations of cryoprotective substances tested were efficient in the cryopreservation of canine MSCs, being alternatives that can be used. All treatments allowed the resumption of normal cell functions after thawing and reculture. And self-formulated solutions are more economically viable than branded. These results are important for the establishment of cryopreservation protocols for MSCs in this species, as they increase the possibilities of choosing the cryoprotectant solution and also allow the use of more affordable solutions without contaminating components such as FBS.
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Palavras-chave
Criobancos, Crioprotetores, Viabilidade, Tecido adiposo