Secretoma de meios condicionados por células-tronco mesenquimais derivadas de tecido adiposo em caninos e felinos produzidos em diferentes ambientes de cultivo
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Data
2019-05-24
Autores
Lara, Maria Laura Lara
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Universidade Estadual Paulista (Unesp)
Resumo
As células-tronco mesenquimais (MSCs) exercem seus efeitos terapêuticos predominantemente pela sua atividade parácrina. Como o secretoma desempenha um papel direto nas atividades biológicas das MSCs, a análise de seus componentes proteicos é um passo fundamental para identificar os principais responsáveis no controle e regulação dos muitos processos biológicos desenvolvidos por essas células. O secretoma de MSCs pode ser modificado e melhorado de forma in vitro para estimular efeitos celulares específicos desejados para aplicações terapêuticas, e uma maneira de modificá-lo é por meio de modificações nas condições de cultura. No presente estudo, objetivou-se descrever o secretoma de células-tronco mesenquimais derivadas de tecido adiposo (AD-MSCs) de gatos e cães submetidos a diferentes condições de cultivo, identificamos e comparamos os efeitos exercidos por diferentes modificações de cultivo. Para a produção de meio condicionado, as células foram descongeladas e cultivadas com meio DMEM/F12 com 20% de FBS, suplementado com antibióticos e antimicóticos em uma incubadora com umidade controlada a 37,5 °C, e 5% de CO2. Após atingir pelo menos 70% de confluência, as garrafas foram lavadas três vezes com solução salina balanceada Hanks (HBSS) e foram cultivadas em quatro condicionamentos distintos. O meio condicionado foi colhido após 4 dias, centrifugado por 10 min/300 g, filtrado em um filtro 0,22 μm e congelado a -80 °C. Foram realizadas a extração e concentração proteica de todas as amostras e depleção de albumina das amostras cultivadas em presença de FBS. A digestão em solução foi realizada para preparar as amostras para a análise por espectrometria de massa. Como resultado, os perfis proteômicos produzidos nas condições de cultura mais estressantes, tiveram uma maior expressão proteica, além de conter a maioria das proteínas determinadas como relevantes. Esse grupo, condicionado pela ausência de FBS e hipóxia 5% O2, apresentou um perfil proteômico voltado à matriz extracelular (ECM) e à modulação da resposta imune, demonstrando a capacidade das MSCs em modular o ambiente de acordo com as circunstâncias apresentadas e tornando-o mais adequado para a proliferação e função celular. Levando em conta que o uso do secretoma como terapia livre de células abre uma nova possibilidade terapêutica para diferentes doenças, é essencial continuar investigando as diferentes modificações de cultivo que podem ser feitas na produção do meio condicionado para direcionar sua utilização a diferentes alvos terapêuticos e diagnósticos.
It is now known that mesenchymal stem cells (MSCs) exert their therapeutic effects predominantly through their paracrine activity. Since the secretome plays a direct role in the biological activities of MSCs, the analysis of their protein components is a fundamental step in identifying the main responsible for the control and regulation of the many biological processes developed by these cells. The MSCs secretome can be modified and improved in vitro to stimulate specific cellular effects desired for therapeutic applications, and one way of modifying it is through modifications in the culture conditions. In the present study, in addition to describing for the first time the secretome of mesenchymal stem cells derived from adipose tissue (AD-MSCs) in feline species, we identified and compared the effects exerted by different culture modifications, such as the use of serum-free medium and hypoxia in the protein production by AD-MSCs in canines and felines. For the conditioned media production, the cells were thawed and cultured with DMEM/F12 medium with 20% fetal bovine serum (FBS), supplemented with antibiotics and antimycotics in controlled incubator ay 37.5 °C with 5% CO2. After reaching at least 70% confluency, the flasks were washed three times with Hanks balanced salt solution (HBSS) and were grown in four different conditions. Conditioned media were collected after 4 days, centrifuged for 10 min/300 g, filtered on a 0.22 μm filter and frozen at -80 °C. Protein extraction and concentration of all samples and albumin depletion of samples cultured with FBS were performed. Digestion in solution also was performed to prepare the samples for mass spectrometry analysis. The proteomic profiles produced under the most stressful culture conditions had a greater protein range, besides containing within it the majority of proteins determined as relevant. This group, conditioned by serum-free medium and 5% hypoxia, presented an extracellular matrix (ECM) and immunomodulation-oriented profile, demonstrating MSCs ability in modulate their environment according to the circumstances and making it more suitable for cell maintenance and function. The secretome as free cell therapy opens new therapeutic possibilities, being essential to continue research of different modifications that can be made in the conditioned medium production to direct its use for different therapeutic and diagnostic targets.
It is now known that mesenchymal stem cells (MSCs) exert their therapeutic effects predominantly through their paracrine activity. Since the secretome plays a direct role in the biological activities of MSCs, the analysis of their protein components is a fundamental step in identifying the main responsible for the control and regulation of the many biological processes developed by these cells. The MSCs secretome can be modified and improved in vitro to stimulate specific cellular effects desired for therapeutic applications, and one way of modifying it is through modifications in the culture conditions. In the present study, in addition to describing for the first time the secretome of mesenchymal stem cells derived from adipose tissue (AD-MSCs) in feline species, we identified and compared the effects exerted by different culture modifications, such as the use of serum-free medium and hypoxia in the protein production by AD-MSCs in canines and felines. For the conditioned media production, the cells were thawed and cultured with DMEM/F12 medium with 20% fetal bovine serum (FBS), supplemented with antibiotics and antimycotics in controlled incubator ay 37.5 °C with 5% CO2. After reaching at least 70% confluency, the flasks were washed three times with Hanks balanced salt solution (HBSS) and were grown in four different conditions. Conditioned media were collected after 4 days, centrifuged for 10 min/300 g, filtered on a 0.22 μm filter and frozen at -80 °C. Protein extraction and concentration of all samples and albumin depletion of samples cultured with FBS were performed. Digestion in solution also was performed to prepare the samples for mass spectrometry analysis. The proteomic profiles produced under the most stressful culture conditions had a greater protein range, besides containing within it the majority of proteins determined as relevant. This group, conditioned by serum-free medium and 5% hypoxia, presented an extracellular matrix (ECM) and immunomodulation-oriented profile, demonstrating MSCs ability in modulate their environment according to the circumstances and making it more suitable for cell maintenance and function. The secretome as free cell therapy opens new therapeutic possibilities, being essential to continue research of different modifications that can be made in the conditioned medium production to direct its use for different therapeutic and diagnostic targets.
Descrição
Palavras-chave
Células mesenquimais estromais, Matriz extracelular, Secretoma, Proteômica, MSCs, Secretome, conditioned medium, proteomic, extracellular matrix