A roadmap towards manufacturing extracellular vesicles for cardiac repair.

For the past two decades researchers have linked extracellular vesicle (EV)-mediated mechanisms to various physiological and pathological processes in the heart, such as immune response regulation, fibrosis, angiogenesis, and the survival and growth of cardiomyocytes. Although use of EVs has gathered momentum in the cardiac field, several obstacles in both upstream and downstream processes during EV manufacture need to be addressed before clinical success can be achieved. Low EV yields obtained in small-scale cultures deter clinical translation, as mass production is a prerequisite to meet therapeutic doses. Moreover, standardizing EV manufacture is critical given the inherent heterogeneity of EVs and the constraints of current isolation techniques. In this review, we discuss the critical steps for the large-scale manufacturing of high-potency EVs for cardiac therapies.

Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) act as signaling mediators of cellular responses. However, despite representing a promising alternative to cell-based therapies, clinical translation of EVs is currently limited by their lack of scalability and standardized bioprocessing. Herein, we integrated scalable downstream processing protocols with standardized expansion of large numbers of viable cells in stirred-tank bioreactors to improve EV production. Higher EV yields were linked to EV isolation by tangential flow filtration followed by size exclusion chromatography, rendering 5 times higher number of EVs comparatively to density gradient ultracentrifugation protocols. Additionally, when compared to static culture, EV manufacture in bioreactors resulted in 2.2 higher yields. Highlighting the role of operating under optimal cell culture conditions to maximize the number of EVs secreted per cell, MSCs cultured at lower glucose concentration favored EV secretion. While offline measurements of metabolites concentration can be performed, in this work, Raman spectroscopy was also applied to continuously track glucose levels in stirred-tank bioreactors, contributing to streamline the selection of optimal EV collection timepoints. Importantly, MSC-derived EVs retained their quality attributes and were able to stimulate angiogenesis in vitro, therefore highlighting their promising therapeutic potential.

Dimethyloxalylglycine, a small molecule, synergistically increases the homing and angiogenic properties of human mesenchymal stromal cells when cultured as 3D spheroids.

Strategies aiming at increasing the survival and paracrine activity of human mesenchymal stromal cells (MSCs) are of utmost importance to achieve the full therapeutic potential of these cells. Herein, we propose both physical and biochemical strategies to enhance the survival, homing, angiogenic, and immunomodulatory properties of MSCs in vitro. To that purpose, we compared the effect of exposing either 2D monolayer or 3D spheroids of MSCs to (i) hypoxia (2% O2 ) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O2 . 3D-cultured MSC spheroids evidenced higher survival upon exposure to oxidative stress and expressed higher levels of factors involved in tissue repair processes, namely tumor necrosis factor-stimulated gene-6, matrix metalloproteinase-2, and vascular endothelial growth factor. MSCs cultured as 3D spheroids and further exposed to hypoxia or hypoxic-mimetic conditions provided by DMOG synergistically favored the expression of the cell surface marker C-X-C chemokine receptor type-4, involved in homing processes to injured tissues, and adhesion to extracellular matrix components as fibronectin. These results highlight the role of ex vivo preconditioning approaches, presenting a novel strategy that combine biochemical stimuli with 3D spheroid organization of MSCs to maximize their tissue regeneration potential.

Three-Dimensional Co-culture of Human Hematopoietic Stem/Progenitor Cells and Mesenchymal Stem/Stromal Cells in a Biomimetic Hematopoietic Niche Microenvironment.

The development of cellular therapies to treat hematological malignancies has motivated researchers to investigate ex vivo culture systems capable of expanding the number of hematopoietic stem/progenitor cells (HSPC) before transplantation. The strategies exploited to achieve relevant cell numbers have relied on culture systems that lack biomimetic niche cues thought to be essential to promote HSPC maintenance and proliferation. Although stromal cells adhered to 2-D surfaces can be used to support the expansion of HSPC ex vivo, culture systems aiming to incorporate cell-cell interactions in a more intricate 3-D environment can better contribute to recapitulate the bone marrow (BM) hematopoietic niche in vitro.Herein, we describe the development of a 3-D co-culture system of human umbilical cord blood (UCB)-derived CD34+ cells and BM mesenchymal stem/stromal cell (MSC) spheroids in a microwell-based platform that allows to attain large numbers of spheroids with uniform sizes. Further comparison with a traditional 2-D co-culture system exploiting the supportive features of feeder layers of MSC is provided, while functional in vitro assays to assess the features of HSPC expanded in the 2-D vs. 3-D MSC co-culture systems are suggested.

Hematopoietic Niche - Exploring Biomimetic Cues to Improve the Functionality of Hematopoietic Stem/Progenitor Cells.

The adult bone marrow (BM) niche is a complex entity where a homeostatic hematopoietic system is maintained through a dynamic crosstalk between different cellular and non-cellular players. Signaling mechanisms triggered by cell-cell, cell-extracellular matrix (ECM), cell-cytokine interactions, and local microenvironment parameters are involved in controlling quiescence, self-renewal, differentiation, and migration of hematopoietic stem/progenitor cells (HSPC). A promising strategy to more efficiently expand HSPC numbers and tune their properties ex vivo is to mimic the hematopoietic niche through integration of adjuvant stromal cells, soluble cues, and/or biomaterial-based approaches in HSPC culture systems. Particularly, mesenchymal stem/stromal cells (MSC), through their paracrine activity or direct contact with HSPC, are thought to be a relevant niche player, positioning HSPC-MSC co-culture as a valuable platform to support the ex vivo expansion of hematopoietic progenitors. To improve the clinical outcome of hematopoietic cell transplantation (HCT), namely when the available HSPC are present in a limited number such is the case of HSPC collected from umbilical cord blood (UCB), ex vivo expansion of HSPC is required without eliminating the long-term repopulating capacity of more primitive HSC. Here, we will focus on depicting the characteristics of co-culture systems, as well as other bioengineering approaches to improve the functionality of HSPC ex vivo.

Tridimensional configurations of human mesenchymal stem/stromal cells to enhance cell paracrine potential towards wound healing processes.

This study proposes to use alginate encapsulation as a strategy to assess the paracrine activity of 3D- and 2D-cultured human bone marrow mesenchymal stem/stromal cells (BM MSC) in the setting of wound repair and regeneration processes. A side-by-side comparison of MSC culture in three different 3D configurations (spheroids, encapsulated spheroids and encapsulated single cells) versus 2D monolayer cell culture is presented. The results reveal enhanced resistance to oxidative stress and paracrine potential of 3D spheroid-organized BM MSC. MSC spheroids (148±2µm diameter) encapsulated in alginate microbeads evidence increased angiogenic and chemotactic potential relatively to encapsulated single cells, as supported by higher secreted levels of angiogenic factors and by functional assays showing the capability of encapsulated MSC to promote formation of tubelike structures and migration of fibroblasts into a wounded area. In addition, a higher expression of the anti-inflammatory factor tumor necrosis factor alpha-induced protein 6 (TSG-6) was demonstrated by RT-PCR for encapsulated and non-encapsulated spheroids. Culture of spheroids within an alginate matrix maintains low aggregation levels below 5% and favors resistance to oxidative stress. These are important factors towards the establishment of more standardized and controlled systems, crucial to explore the paracrine effects of 3D-cultured MSC in therapeutic settings.