Evaluation of Taylor vortex flow bioreactor performance for mesenchymal stromal cell expansion

Interest in the use of mesenchymal stromal cells (MSCs) has increased in the last 10 years because of the increasing discovery of its special characteristics for the treatment of diseases through cell therapy or tissue engineering. Despite being in the majority of postnatal organs and tissues, the amount of MSCs required to initiate any clinical treatment is insufficient and therefore the necessity of its in vitro expansion. Cultivation systems with the use of microcarriers and bioreactors, such as the stirred tank, are presented as the immediate proposal, allowing to obtain high cellular densities in a 3D environment. However, the stirred tank presents some limitations related to the distribution of hydrodynamic forces within the bioreactor and the difficulty in controlling the size of aggregates formed during cultivation, ultimately affecting the process yield of obtaining viable cells. A highly promising bioreactor proposal for presenting low and more homogeneous shear stresses, satisfactory mass transfer and ease of scaling is the Taylor Vortex Flow Bioreactor (TVFB). In a preliminary study, the BEVT, with a work volume of 60 mL, was promising in the cultivation of a line of immortalized CEMs attached to microcarriers, presenting controlled sizes of cellular aggregates (<400 μm) under conditions of low shear stresses and with sufficient oxygen transfer. In this sense, the present work involved the construction and the study of a BEVT with a work volume of 100 mL, for the cultivation of primary CEMs currently used in clinical treatments. Thus, the additional evaluation of important hydrodynamic parameters, such as the minimum velocity of suspension of particles (Ns1) and time of mixing (θm) in different conditions of rotation, were approached for two internal cylinder configurations, named in this study as round and deformed. In this evaluation the BEVT with the deformed inner cylinder presented better performance and was chosen in the culture studies. For this configuration Ns1 values between 70 and 90 rpm were obtained using a concentration of 1 to 3 g of microcarregages/mL and a maximum θm of 21±2,2 seconds for a rotation speed of 50 rpm. Cultivation of umbilical cord derived CEMs cultures and Cultispher®S 1 and 3 g / L adipose tissue using the 10% SFB alpha MEM culture medium confirmed the best modulation of the aggregation presented in the BEVT, compared to the Spinner flask, under low shear conditions, confirmed by the LDH enzyme activity, and with adequate oxygen transfer, obtaining factors of expansion of up to 17 times of viable and functional CEMs. Thus, these results present the BEVT as a very promising alternative for the cultivation of large-scale CEMs for cell therapy.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)O interesse no uso das células estromais mesenquimais (CEMs) tem aumentado nos últimos 10 anos como consequência do crescente descobrimento de suas características atraentes na regeneração de órgão e tecido, no campo da terapia celular e engenharia de tecidos. Apesar de estar presente na maioria dos órgãos e tecidos pós-natais, a quantidade de CEMs requeridas para iniciar qualquer tratamento clínico é insuficiente e por isso há necessidade de sua expansão in vitro. Sistemas de cultivo com o uso de microcarregadores e biorreatores, como o tradicional tanque agitado, são apresentados como a proposta mais imediata, permitindo obter altas densidades celulares em um ambiente 3D mais parecido com o in vivo. Apesar do tanque agitado ser o mais utilizado em vários bioprocessos, em cultivo de células aderentes tem apresentado algumas limitações relacionadas com a distribuição heterogênea de forças hidrodinâmicas dentro do biorreator e a dificuldade para controlar o tamanho de agregados formados durante o cultivo, afetando finalmente o rendimento do processo de obtenção de células viáveis e funcionais necessárias nos tratamentos clínicos. Uma proposta bastante promissora de biorreator por apresentar baixas e mais homogêneas tensões de cisalhamento, transferência de oxigênio satisfatória e facilidade no escalonamento é o Biorreator de Escoamento em Vórtices de Taylor (BEVT). Em um estudo preliminar, o BEVT, com um volume de trabalho de 60 mL, se mostrou promissório no cultivo de uma linhagem de CEMs imortalizadas aderidas a microcarregadores, apresentando tamanhos controlados de agregados celulares (