Expansão de células-tronco mesenquimais em biorreator single-use com movimento ondulatório visando aplicações terapêuticas

Abstract

Mesenchymal Stem/Stromal Cells (MSCs) and MSC-derived Extracellular Vesicles (EVs) are being targeted in different medical areas. The MSCs are present in small proportions in tissues, thus making an in vitro expansion necessary, which allows the therapeutic applicability. The single-use bioreactor technology, such as the Wave-Induced Motion Bioreactor (WIMB), is attractive due to the possibility of controlled and pre-sterilized system operation. However, the unidirectional wave motion leads to the formation of particle deposits (microcarriers and cells) on the Cellbag. The formation of the “dune-like” deposits and the deposits at the Cellbag wall (border region of the liquid) is facilitated by the increase in the hydrophilicity of the Cellbag inner film during culture. This problem impairs the MSC expansion, leading to cell expansion factors (EF) lower than 2.0. The objective of this work was to improve the Wave BioreactorTM 2/10 performance in the adhesion and expansion of Human Umbilical Cord Matrix derived Mesenchymal Stem/Stromal Cells (hUCM-MSCs) and to evaluate the cultures with respect to the expansion potential and the hUCM-MSCs therapeutic profile. The Cellbag design was modified by the implementation of a grid designed in our laboratory and placed on the platform, aiming to reduce the microcarrier deposits. Cultures were performed using CultiSpher-S® and MAG microcarriers with α-MEM culture media (10% v/v FBS). Two approaches were evaluated with the new Cellbag design: 1) adhesion phase in spinner and expansion in the WIMB with CultiSpher-S® (EF: 7.3±1.1 fold) and 2) adhesion and expansion phases in the WIMB with CultiSpher-S® (EF between 6.3 and 25.6 fold) and MAG (EF: 5.5±0.6 fold). The hUCM-MSCs harvested from the WIMB cultures showed the quality attributes. The control cultures in spinner with MAG and CultiSpher-S® and the WIMB cultures with CultiSpher-S® showed upregulated genes linked to biological functions of cell adhesion, inflammatory response, immune response, extracellular matrix organization and collagen synthesis, in relation to the monolayer-static culture. The WIMB cultures produced, on average, 11 times more EVs per hUCM-MSC when compared to the spinner culture. These results showed that the Cellbag design modification inhibited the formation of "dune-like" deposits, which improved the WIMB performance, making possible the use of wave-induced motion bioreactors in stem cell bioprocessing.