Nieuwland, Rienk

ABSTRACT The FDA Modernization Act 3.0 represents a pivotal shift in biomedical research by formally removing the requirement for animal testing and enabling regulatory acceptance of New Approach Methodologies (NAMs). This transition creates an urgent need for robust, human‐relevant biomarkers that can anchor NAM‐generated data within regulatory decision‐making, particularly for first‐in‐human studies. Extracellular vesicles (EVs), owing to their biological stability, accessibility and rich molecular cargo, may be well positioned to fulfil this role. EVs reflect dynamic cellular states and intercellular communication, making them mechanistically informative readouts of pharmacology, toxicity, immune modulation and disease biology across human‐derived model systems. This Perspective outlines how EV‐based biomarkers can support the qualification, standardization, and regulatory acceptance of NAMs, with particular emphasis on ex vivo platforms such as organoids, microtissue systems, organ‐on‐chip devices and perfused tissue slices. We highlight the advantages of EVs for longitudinal, non‐destructive sampling, representation of human variability, and integration with quantitative systems pharmacology and physiologically based pharmacokinetic models. Finally, we discuss the scientific, operational and regulatory challenges that must be addressed and argue that coordinated leadership from the International Society for Extracellular Vesicles (ISEV) is essential. The convergence of regulatory reform and EV science presents a timely call to action to establish EVs as foundational biomarkers in next‐generation, human‐centric drug development.

ABSTRACT The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell‐derived EVs using flow cytometry. To address the orders‐of‐magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %–31 %. The calibration methodology was then used to determine reference ranges of erythrocyte‐, leukocyte‐, and platelet‐derived EVs in human blood plasma in a cohort of healthy individuals ( n = 224). This study demonstrates that calibration enables comparable concentration measurements of blood cell‐derived EVs, thereby bringing EVs one step closer to clinical applications.

Abstract Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Abstract Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor‐dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre‐analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood‐EV), a tool to record and report information about pre‐analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre‐analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence‐based optimization of pre‐analytical protocols through meta‐analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter‐laboratory comparative EV studies, and (iv) improve the peer review process.