Speaker
Description
Introduction
The secretion of therapeutic factors and extracellular vesicles (EVs) by mesenchymal stromal cells (MSCs) gained much interest for their benefits in the regenerative medicine field. Indeed, the in vitro multipotency of these cells and the secretion of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture in monolayer, MSCs rapidly lose the expression of key transcription factors associated
with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In this scenario, it is essential to find alternative culture methods, which more faithfully simulate the physiological characteristics of these cells. It has been reported that 3D cultures allow cells to be cultured for a longer period, without losing their stemness, and mimicking the situation that occurs in vivo. The aim of this work was to evaluate if 3D culture can determine
also an effect on EVs secretion.
Methodology
MSCs were obtained from bone marrow aspirates. Cells were cultured until passage 2, then spheroids were created using the 3D Petri Dish® system. It consists of a tiny 96-well plate of 2% agar, which does not allow the cells to adhere, stimulating their aggregation. Cell were also cultured in monolayer as control. Cell in both conditions were analysed by real-time PCR and flow cytometry. EVs were also isolated from conditioned media. They were characterized through different methods including nanoparticle tracking analysis, western blot, non- conventional flow cytometry, according to MISEV 2018.
Results
Spheroid cultures from MSCs showed a drastic decrease in dimension after first days in culture, due to a cytoskeleton reorganization which led to a morphology change. Cells in 3D lose the classical fibroblastic spindle shape morphology, becoming polygonal. Interestingly, despite maintaining the stem characteristics, MSCs grown in 3D partially modify their surface phenotype, expressing CD31 and CD34. These are surface markers normally negative in mesenchymal lineage and positive in hematopoietic cells. Analysing the EVs released by MSCs cultured in 2D and 3D, we observed that spheroid cultures exhibit a higher release of EVs, however maintaining similar characteristics to the EVs derived from MSCs in 2D.
Conclusions
The use of this culture strategy could be a valid alternative to traditional culture methods for EVs production by MSCs. As future perspective, a dynamic system could be used in order to better mimic the in vivo environment.
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