Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources (bone marrow (BM), adipose tissue (AT) and umbilical cord matrix (UCM)). A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-Wheel™ bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGRO™-PURE), towards the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between -15.5 ± 1.6 mV and - 19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e. EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e. amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 x 10^11, 3.1 ± 1.3 x 10^11 and 4.1 ± 1.7 x 10^11 EV particles (n=3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, towards the development of novel therapeutic products.

Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT⁺ cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-β/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.

Neural stem cells (NSCs) have the potential to generate the cells of the nervous system and, when cultured on nanofiber scaffolds, constitute a promising approach for neural tissue engineering. In this work, the impact of combining nanofiber alignment with functionalization of the electrospun poly-ε-caprolactone (PCL) nanofibers with biological adhesion motifs on the culture of an NSC line (CGR8-NS) is evaluated. A five-rank scale for fiber density was introduced, and a 4.5 level, corresponding to 70–80% fiber density, was selected for NSC in vitro culture. Aligned nanofibers directed NSC distribution and, especially in the presence of laminin (PCL-LN) and the RGD-containing peptide GRGDSP (PCL-RGD), promoted higher cell elongation, quantified by the eccentricity and axis ratio. In situ differentiation resulted in relatively higher percentage of cells expressing Tuj1 in PCL-LN, as well as significantly longer neurite development (41.1 ± 1.0 μm) than PCL-RGD (32.0 ± 1.0 μm), pristine PCL (25.1 ± 1.2 μm), or PCL-RGD randomly oriented fibers (26.5 ± 1.4 μm), suggesting that the presence of LN enhances neuronal differentiation. This study demonstrates that aligned nanofibers, functionalized with RGD, perform as well as PCL-LN fibers in terms of cell adhesion and proliferation. The presence of the full LN protein improves neuronal differentiation outcomes, which may be important for the use of this system in tissue engineering applications.