Scalability of spheroid-derived small extracellular vesicles production in stirred systems

IntroductionSmall extracellular vesicle (sEV)-based therapies have gained widespread interest, but challenges persist to ensure standardization and high-scale production. Implementing upstream processes in a chemically defined media in stirred-tank bioreactors (STBr) is mandatory to closely control the cell environment, and to scale-up production, but it remains a significant challenge for anchorage-dependent cells.MethodsWe used a human β cell line, grown as monolayer or in suspension as spheroid in stirred systems. We assessed the consequences of culturing these cells in 3D with, or without fetal bovine serum in a chemically defined medium, for cell growth, viability and metabolism. We next explored how different scale-up strategies might influence cell and spheroid formation in spinner flask, with the aim to transfer the process in instrumented Ambr®250 STBr. Lastly, we analyzed and characterized sEV production in monolayer, spinner flask and STBr.Results and discussionGeneration of spheroids in a chemically defined medium allowed the culture of highly viable cells in suspension in stirred systems. Spheroid size depended on the system’s volumetric power input (P/V), and maintaining this parameter constant during scale-up proved to be the optimal strategy for standardizing the process. However, transferring the spinner flask (SpF) process to the Ambr®250 STBr at constant P/V modified spheroid size, due to important geometric differences and impeller design. Compared to a monolayer reference process, sEV yield decreased two-fold in SpF, but increased two-fold in STBr. Additionally, a lower expression of the CD63 tetraspanin was observed in sEV produced in both stirred systems, suggesting a reduced release of exosomes compared to ectosomes. This study addresses the main issues encountered in spheroid culture scale-up in stirred systems, rather conducive for the production of ectosomes

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Bioproduction de vésicules extracellulaires à partir de cultures de sphéroidess de cellules humaines en bioréacteurs à cuves agitées

Extracellular vesicles (EV) are a promising new class of biotherapies, but challenges remain regarding their standardized and large-scale production. The implementation of production in stirred tank bioreactors (STBr) is the preferred solution to finely control the cellular environment and facilitate process scale-up. However, this represents a major challenge for adherent cells. β cell-derived EV are of particular interest in the context of type 1 diabetes therapy. In this work, we show that spheroid formation in a chemically defined medium enables the culture of highly viable 1.4E7 cells in STBr. . The size and concentration of the spheroids depend on the system's power input per unit volume, and maintaining this parameter constant during scale-up allows for standardized spheroid formation in geometrically similar system. Compared to the reference monolayer process, the EV yield doubles in STBr. Additionally, the reduced expression of CD63 in EV produced in STBr suggests an enrichment in ectosomes, while a distinct cytokine profile was observed during mixed leukocyte reactions, compared to monolayer-derived EV. This thesis explores the opportunities presented by EV production from spheroid cultures in STBr, while addressing the limitations of this approach and future perspectives for overcoming them.Les vésicules extracellulaires (EV) sont une nouvelle classe de biothérapies prometteuses, mais des défis subsistent quant à leur production standardisée et à grande échelle. La mise en place de productions en bioréacteurs à cuve agitée (STBr) est la solution privilégiée pour contrôler finement l’environnement cellulaire et faciliter l'extrapolation du procédé. Toutefois, cela représente un défi majeur pour les cellules adhérentes. Les EV de cellules β représentent notamment un intérêt dans le cadre de la thérapie du diabète de type 1. Dans ce travail, nous montrons que la formation de sphéroïdes en milieu chimiquement défini permet de cultiver des cellules 1.4E7 hautement viables en STBr. La taille et la concentration des sphéroïdes dépendent de la puissance volumique du système, et maintenir ce paramètre constant lors de l’extrapolation permet de standardiser leur formation dans des systèmes à géométrie similaire. Comparé au procédé de référence en monocouche, le rendement en EV double en STBr. De plus, l'expression réduite de CD63 dans les EV produites en STBr suggère un enrichissement en ectosomes, tandis qu'un profil de cytokines différent a été observé lors de réactions leucocytaires mixtes, par rapport aux EV produites en monocouches. Ce travail de thèse explore les opportunités offertes par la production d'EV à partir de cultures de sphéroïdes en STBr, tout en abordant les limites de cette approche et les perspectives pour y remédier

Bioproduction of extracellular vesicles from spheroid cultures of human cells in stirred-tank bioreactors

Les vésicules extracellulaires (EV) sont une nouvelle classe de biothérapies prometteuses, mais des défis subsistent quant à leur production standardisée et à grande échelle. La mise en place de productions en bioréacteurs à cuve agitée (STBr) est la solution privilégiée pour contrôler finement l’environnement cellulaire et faciliter l'extrapolation du procédé. Toutefois, cela représente un défi majeur pour les cellules adhérentes. Les EV de cellules β représentent notamment un intérêt dans le cadre de la thérapie du diabète de type 1. Dans ce travail, nous montrons que la formation de sphéroïdes en milieu chimiquement défini permet de cultiver des cellules 1.4E7 hautement viables en STBr. La taille et la concentration des sphéroïdes dépendent de la puissance volumique du système, et maintenir ce paramètre constant lors de l’extrapolation permet de standardiser leur formation dans des systèmes à géométrie similaire. Comparé au procédé de référence en monocouche, le rendement en EV double en STBr. De plus, l'expression réduite de CD63 dans les EV produites en STBr suggère un enrichissement en ectosomes, tandis qu'un profil de cytokines différent a été observé lors de réactions leucocytaires mixtes, par rapport aux EV produites en monocouches. Ce travail de thèse explore les opportunités offertes par la production d'EV à partir de cultures de sphéroïdes en STBr, tout en abordant les limites de cette approche et les perspectives pour y remédier.Extracellular vesicles (EV) are a promising new class of biotherapies, but challenges remain regarding their standardized and large-scale production. The implementation of production in stirred tank bioreactors (STBr) is the preferred solution to finely control the cellular environment and facilitate process scale-up. However, this represents a major challenge for adherent cells. β cell-derived EV are of particular interest in the context of type 1 diabetes therapy. In this work, we show that spheroid formation in a chemically defined medium enables the culture of highly viable 1.4E7 cells in STBr. The size and concentration of the spheroids depend on the system's power input per unit volume,and maintaining this parameter constant during scale-up allows for standardized spheroid formation in geometrically similar system. Compared to the reference monolayer process, the EV yield doubles in STBr. Additionally, the reduced expression of CD63 in EV produced in STBr suggests an enrichment in ectosomes, while a distinct cytokine profile was observed during mixed leukocyte reactions, compared to monolayer-derived EV. This thesis explores the opportunities presented by EV production from spheroid cultures in STBr, while addressing the limitations of this approach and future perspectives for overcoming them

Unlocking the potential of bio-inspired bioinks: A collective breakthrough in mammalian tissue bioprinting

International audienceThe composition of soft tissues in mammals can be simplified as approximately 60–65 % water, 16 % protein, 16 % fat, 1 % carbohydrate, and trillions of cells. This report brings together unpublished results from a collaborative efforts of 10 research groups over the past five years, all dedicated to producing mammalian tissues through extrusion-based bioprinting. What unified these studies was a common approach, with a shared bioink composition consisting of gelatin, alginate, and fibrinogen, and a post-printing consolidation strategy involving transglutaminase crosslinking, calcium chelation, and thrombin-mediated fibrin production. The range of Young’s moduli achievable was 0.17–105 kPa, perfectly align with of tissue properties.By consolidating the findings of these studies, it was conclusively demonstrated that bioprinting and culturing all 19 cells tested from 14 different organs was indeed achievable. These remarkable outcomes were attributed not only to the bio-inspired nature of the common bioink but also to its unique rheological properties, such as significant shear-thinning and a sufficiently high static yield stress.The majority of these cells exhibited behaviours consistent with their natural in vivo environments. Clearly identifiable microstructures and organizations showcased intricate morphogenesis mechanisms resulting in the formation of micro-tubules, micro-vessels, and micro-acini. It is now evident that microextrusion bioprinting, especially when using bio-inspired bioink formulations, represents a promising avenue for generating a wide range of mammalian soft tissues

Phenotype and Outcomes of Pulmonary Hypertension Associated with Neurofibromatosis Type 1

International audienc

Unlocking the potential of bio-inspired bioinks: A collective breakthrough in mammalian tissue bioprinting

International audienc