Welcome and Introduction to the Conference by Conference Chairperson
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Nanospresso: Microfluidic Mixing of Nucleic Acids and Excipients to Make Nanomedicines
Raymond Schiffelers, Professor of Nanomedicine, University Medical Center Utrecht, Netherlands

Lipid nanoparticles are the frontrunner nanomedicines for the delivery of nucleic acid therapeutics. The COVID-19 mRNA vaccines were built on this technology. These lipid nanoparticles self-assemble around the mRNA through electrostatic and hydrophobic interactions. For mass production of lipid nanoparticles (like vaccines during a pandemic) impingement jet technology provides high throughput. For smaller batches, microfluidic mixing can provide an excellent platform for making high quality lipid nanoparticles. In the project NANOSPRESSO, we take this concept one step further to explore microfluidic mixing of personalized lipid nanoparticles in a cartridge for a single dose for a single patient produced in the hospital pharmacy. This concept offers attractive properties for the democratization of personalized healthcare.

EV MEET Cell: An Efficient EV-Target Cell Interaction through Modulation of the Extracellular Environment of Target cell
Sai Kiang Lim, Research Director, Institute of Medical Biology, A*STAR, Singapore

Mesenchymal stromal cells (MSC) have displayed promising therapeutic potential; nonetheless, no FDA-approved MSC product exists due largely to the absence of a reliable potency assay based on the mechanisms of action to ensure consistent efficacy. MSCs are now thought to exert their effects primarily by releasing small extracellular vesicles (sEVs) of 50-200 nm. While non-living MSC-sEV drugs offer distinct advantages over larger, living MSC drugs, elucidating their mechanism of action to develop robust potency assays remains a challenge. A pivotal prelude to elucidating the mechanism of action for MSC-sEV is how extracellular vesicles (EVs) communicate with their primary target cells. Given the inherent inefficiencies of processes like endocytosis, endosomal escape, and EV uncoating during cellular internalization, we propose an alternative EV communication approach, EV-MEET Cell, which stands for EV-Modulation of the Extracellular Environment of Target Cells. This approach involves modifying the external cellular milieu such that signals from a single EV could be transmitted to multiple recipient cells, thereby eliciting a more widespread tissue response.

EV-based Therapeutics Development: No Place for the Weak
Mario Gimona, Head of Manufacturing, GMP Unit, Paracelsus Medical University Salzburg, Austria

Extracellular vesicle (EV)-enriched preparations obtained from multipotent mesenchymal stromal cells (MSCs) are of particular interest for therapeutic use since they may convey anti-inflammatory, anti-fibrotic and cyto-protective activities to the target tissues. MSCs secrete a variety of bioactive autocrine and paracrine factors including cytokines, chemokines, extracellular matrix proteins, EVs and growth factors. The circulating MSC-derived secretome is thus a complex mixture of proteins, lipids, macromolecular assemblies and vesicular structures, including various types of EVs. Current activities aim at quantitatively describing one or more biological activities present in/on EVs that are beneficial in elucidating the complex mechanism-of-action of the vesicular secretome, and that can assist in the required dose-finding for pre-clinical and clinical application. Understanding EV composition and dynamics will further increase the value of these nanosized particles as a potential tool for companion diagnostics.

ExoPulse™ Production Platform: 1st, 2nd and 3rd Generation EV
Marcin Jurga, Chief Scientific Officer, EXO Biologics SA, Belgium

NanoFACS Isolated Placental Cell- and Size-Specific EVs and T-cell Polarization
Terry Morgan, Professor of Pathology and Laboratory Medicine, Oregon Health and Science University, United States of America

How the placenta suppresses the maternal immune response to this invasive fetal organ is a long-standing mystery. New data suggest that placental extracellular vesicles (EVs) isolated from first trimester placental explants in vitro induce immune tolerance (ie; Bai K et al, 2022).  This is similar to Treg induction by cancer EVs, but shares the method-based weakness of testing heterologous fractions of EVs from many different cell sources.  A significant advance would be to isolate placental cell- and size-specific EVs and culture them with T-cells to determine the dose-response effect. Moreover, an important negative control would be to test isolated liposomes from the same plasmas.  In this study, we hypothesize that placental invasive extravillous trophoblast EVs induce T-cell polarization from Th1 to Tregs compared with liposomes isolated from the same plasma samples.  We isolated placental EVs and spiked-in 200nm liposomes by nanoFACS from banked human first trimester plasma samples.  T-cell polarization cultures from banked PBMCs (positive control was IL-2 induced +/- TGF-b/retinoic acid) were validated and then compared with banked PBMCs treated with either placental EVs or liposomes. Our results show that placental EVs have PD-L1 on their surfac and compared with liposomes incubated with Th1 cells show significantly more Treg and Th2 polarization after 3 days in culture.

Recent Developments in IVDR, Including LDTs
Erik Vollebregt, Partner, Axon Lawyers, Netherlands

• Recent developments in IVDR
• Developments in IVDR regulation of in-house developed devices / LDTs
• Another move of deadlines per risk class
• New obligations for manufacturers to inform of supply interruptions and discontinuations

Integrating Microphysiological Systems and Extracellular Vesicle-Based Technologies to Advance Regenerative Medicine
Danilo Tagle, Director, Office of Special Initiatives, National Center for Advancing Translational Sciences at the NIH (NCATS), United States of America

Microphysiological systems are microfluidic cell culture chips capable of recapitulating key functional aspects of physiological human tissue and organ response. MPS have many contexts of use including evaluation of toxicity/safety, and efficacy of promising therapeutic compounds, disease modeling of both rare and common diseases, as well as within the regenerative and precision medicine space. Extracellular vesicles (EVs) are nano-sized, membrane-enclosed carriers of bioactive lipids, protein, and nucleic acids that are used for intracellular communication. Extracellular vesicles (EVs), membrane-bound particles containing a variety of RNA types, DNA, proteins and other macromolecules, are now appreciated as an important means of communication between cells and tissues, both in normal cellular physiology and as a potential indicator of cellular stress and other environmental exposures and early disease pathogenesis. EVs have pleiotropic actions in physiological and pathological conditions.  EVs are commonly heterogeneous in size, ranging from 20 to 1,000 nm in diameter depending on their origin and mechanism of release, direct shedding or budding from the plasma membrane. Exosomes are vesicles with a diameter of 20–100 nm formed by the inward budding of endosomal membranes to form large multivesicular bodies (MVBs) and released extracellularly when MVBs fuse with the plasma membrane.  Exosomes have recently been studied for their potential use in therapy as a 1) targeted and non-immunogenic delivery system for drugs or biological molecules, and 2) in the maintenance of tissue homeostasis and their contribution to tissue repair and regeneration. For the past few years, MPS and EV-based technologies have been combined within the regenerative medicine space to find safer, more efficacious patient therapies, as well as to probe for non-invasive diagnostic biomarkers. Combination of these technologies could potentially help address a key drug development challenge, i.e., on-target delivery without off-tissue toxicity by delivering therapeutics (small molecules, macromolecules, nucleic acids, etc.) via EVs that only act at the diseased tissue, regardless of whether a target is expressed elsewhere. This presentation will summarize NIH-funded activities in exploring the therapeutic applications of exosomes along with application of new experimental models, including organ-on-chip (OOC) systems and in vitro approaches to extend findings.