Periodic Reporting for period 1 - EVoid (High-Throughput Production of Extracellular Vesicles from Organoids under Rotating Motion)
Reporting period: 2024-10-01 to 2026-03-31
Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, have emerged as central mediators of intercellular communication, with key roles in physiological homeostasis and major diseases such as cancer and regenerative disorders. Their unique advantages over cell-based therapies, particularly in terms of stability, storage, and safety, have driven a rapid expansion of clinical interest. However, the development of EV-based therapeutics has been critically hindered by the lack of robust, scalable, and high-yield production technologies. Conventional approaches rely on low endogenous secretion rates or modest induction strategies, resulting in limited yields, long production times, and high material costs. Moreover, existing systems are not adapted to advanced and physiologically relevant cellular models, such as organoids, despite clear evidence that the therapeutic identity and functional properties of EVs are strongly dependent on the state and environment of their parental cells. This is particularly limiting in the context of pluripotent stem cells, which are becoming a cornerstone of next-generation therapies but remain largely underexploited for EV production, especially in organoid configurations.
Within this context, EVoid has addressed these critical technological and conceptual bottlenecks by establishing a novel paradigm for EV bioproduction. The project demonstrated the feasibility of integrating the formation of physiologically relevant 3D cellular constructs, spheroids and organoids, with high-throughput EV production in a single hydrodynamic platform. This approach enabled a significant increase in production yield while reducing processing time and cell material requirements, and simultaneously improved the biological relevance and specificity of the produced EVs. By bridging advanced cell culture systems with scalable biomanufacturing, EVoid provides a coherent response to the unmet need for efficient and versatile EV production technologies. The results position this technology as a potential enabler for the industrialisation of EV-based therapies and for the development of next-generation biologics with enhanced functional properties, thereby contributing to the broader advancement of regenerative medicine, precision therapeutics, and biomedical innovation.
In addition, the project contributed to the validation of a novel EV characterisation technique combining interferometric scattering and holographic tracking, enabling highly sensitive detection of very small particles together with quantitative measurements of size, scattering properties, and concentration.
Within this context, EVoid has addressed these critical technological and conceptual bottlenecks by establishing a novel paradigm for EV bioproduction. The project demonstrated the feasibility of integrating the formation of physiologically relevant 3D cellular constructs, spheroids and organoids, with high-throughput EV production in a single hydrodynamic platform. This approach enabled a significant increase in production yield while reducing processing time and cell material requirements, and simultaneously improved the biological relevance and specificity of the produced EVs. By bridging advanced cell culture systems with scalable biomanufacturing, EVoid provides a coherent response to the unmet need for efficient and versatile EV production technologies. The results position this technology as a potential enabler for the industrialisation of EV-based therapies and for the development of next-generation biologics with enhanced functional properties, thereby contributing to the broader advancement of regenerative medicine, precision therapeutics, and biomedical innovation.
In addition, the project contributed to the validation of a novel EV characterisation technique combining interferometric scattering and holographic tracking, enabling highly sensitive detection of very small particles together with quantitative measurements of size, scattering properties, and concentration.
During the EVoid project, the work focused on overcoming two major and previously unaddressed challenges EV bioproduction: the need for physiologically relevant production environments and the achievement of high, scalable production yields. To this end, a novel hydrodynamic bioreactor technology based on a baffled tube operating under alternating rotation was developed, implemented, and validated. A first functional prototype at the 10–100 mL scale was engineered, integrating dedicated hardware (motors and microcontrollers) with in-house control software, enabling precise tuning of hydrodynamic conditions.
Using this platform, the project demonstrated the ability to simultaneously induce cell aggregation and stimulate EV production. Human mesenchymal stem cells (hMSCs) were successfully cultured as spheroids directly within the device and used as EV-producing units. The resulting EVs displayed preserved morphology and significantly enhanced secretion rates, reaching up to 20-fold higher yields compared to conventional static culture methods. Beyond yield, EVoid established that hydrodynamic stimulation directly impacts EV composition and quality. Produced EVs exhibited increased total RNA content and a distinct proteomic profile, including enrichment in mitochondrial and microvesicle-associated proteins, indicating enhanced functional potential. Furthermore, the process improved batch-to-batch homogeneity, addressing a critical requirement for reproducibility and future clinical translation. The technological platform was further extended to more complex and physiologically relevant models, including iPSC-derived organoids.
In parallel, the underlying hydrodynamic principles were systematically investigated, demonstrating the key roles of internal baffles, rotational regimes, and flow reversal in controlling turbulence, shear stress, and production scalability.
The versatility of the approach was additionally demonstrated through its successful application to bacterial EV production, achieving similarly high yields and distinct cargo profiles. Moreover, the project established that hydrodynamic stimulation can be tuned to modulate biological outputs beyond EVs, including enhanced RNA loading and the production of lentiviral and virus-like particles.
The main outcomes of the action include: (i) the validation of a disruptive bioproduction technology enabling high-yield and scalable EV production in physiologically relevant 3D systems; (ii) the demonstration of improved EV quality, consistency, and functional potential; (iii) the extension of the technology to multiple biological systems and particle types; and (iv) the consolidation of the scientific and technological foundations required for further development toward industrial and clinical applications.
In addition, EVoid contributed to the development of a novel label-free technique for the characterisation and quantification of ultra-small EVs, demonstrating performance beyond conventional particle tracking, dynamic light scattering, and interferometric methods.
Using this platform, the project demonstrated the ability to simultaneously induce cell aggregation and stimulate EV production. Human mesenchymal stem cells (hMSCs) were successfully cultured as spheroids directly within the device and used as EV-producing units. The resulting EVs displayed preserved morphology and significantly enhanced secretion rates, reaching up to 20-fold higher yields compared to conventional static culture methods. Beyond yield, EVoid established that hydrodynamic stimulation directly impacts EV composition and quality. Produced EVs exhibited increased total RNA content and a distinct proteomic profile, including enrichment in mitochondrial and microvesicle-associated proteins, indicating enhanced functional potential. Furthermore, the process improved batch-to-batch homogeneity, addressing a critical requirement for reproducibility and future clinical translation. The technological platform was further extended to more complex and physiologically relevant models, including iPSC-derived organoids.
In parallel, the underlying hydrodynamic principles were systematically investigated, demonstrating the key roles of internal baffles, rotational regimes, and flow reversal in controlling turbulence, shear stress, and production scalability.
The versatility of the approach was additionally demonstrated through its successful application to bacterial EV production, achieving similarly high yields and distinct cargo profiles. Moreover, the project established that hydrodynamic stimulation can be tuned to modulate biological outputs beyond EVs, including enhanced RNA loading and the production of lentiviral and virus-like particles.
The main outcomes of the action include: (i) the validation of a disruptive bioproduction technology enabling high-yield and scalable EV production in physiologically relevant 3D systems; (ii) the demonstration of improved EV quality, consistency, and functional potential; (iii) the extension of the technology to multiple biological systems and particle types; and (iv) the consolidation of the scientific and technological foundations required for further development toward industrial and clinical applications.
In addition, EVoid contributed to the development of a novel label-free technique for the characterisation and quantification of ultra-small EVs, demonstrating performance beyond conventional particle tracking, dynamic light scattering, and interferometric methods.
Compared to the state of the art, which relies either on low-yield spontaneous EV secretion or on artificial substrate-based systems that are poorly suited to advanced cell models, EVoid introduces a fundamentally different paradigm by combining substrate-free 3D culture (spheroids and organoids) with controlled hydrodynamic stimulation. This enables EV production under physiologically relevant conditions while achieving unprecedented secretion efficiencies, ranging from approximately 2- to 20-fold higher than existing approaches. Importantly, the system also ensures improved batch-to-batch reproducibility through shortened and standardised production cycles, addressing a major limitation of current EV manufacturing technologies. In addition, hydrodynamic triggering not only increases yield but also modulates EV molecular composition, resulting in vesicles with enhanced therapeutic signatures.
Beyond its scientific advances, EVoid establishes a new technological foundation for scalable, high-quality EV production compatible with clinically relevant cell sources such as hMSC-derived spheroids and iPSC-derived organoids. Its main expected impact lies in enabling the transition of EV-based therapies from experimental settings to industrial and clinical-scale production, by providing a platform that integrates cell engineering, organoid maturation, and EV harvesting in a single system. Key conditions for further uptake include continued technological development toward industrial scale-up, regulatory alignment for clinical-grade manufacturing, and support for translation through IP protection and industrial partnerships. Overall, EVoid positions itself as a potential reference technology for next-generation EV biomanufacturing, addressing a rapidly expanding market and a critical bottleneck in regenerative and precision medicine.
Beyond its scientific advances, EVoid establishes a new technological foundation for scalable, high-quality EV production compatible with clinically relevant cell sources such as hMSC-derived spheroids and iPSC-derived organoids. Its main expected impact lies in enabling the transition of EV-based therapies from experimental settings to industrial and clinical-scale production, by providing a platform that integrates cell engineering, organoid maturation, and EV harvesting in a single system. Key conditions for further uptake include continued technological development toward industrial scale-up, regulatory alignment for clinical-grade manufacturing, and support for translation through IP protection and industrial partnerships. Overall, EVoid positions itself as a potential reference technology for next-generation EV biomanufacturing, addressing a rapidly expanding market and a critical bottleneck in regenerative and precision medicine.