Overview and intent

Extracellular vesicles (EVs) and viral particles were enriched onboard from CTD-rosette seawater to generate a clean, concentrated fraction suitable for downstream particle characterization (e.g., NTA), imaging (e.g., EM), and molecular assays. For each sampled depth, 100 L of seawater were processed the same day of collection and reduced to a final 1 mL EV/virus-enriched eluate, which was then subdivided into 10 × 100 µL aliquots, snap-frozen in liquid nitrogen, and stored at −80 °C.

Sample collection and handling

Seawater was obtained from Niskin bottles mounted on a CTD rosette. For each depth, the targeted volume (100 L) was transferred into cleaned, clearly labeled carboys. From the moment of collection, the sample was handled as a cold, light-protected matrix: carboys were kept in cold, dark conditions and moved promptly into processing to minimize changes in particle integrity and community composition before enrichment.

Sequential pre-filtration to remove larger material

To reduce the burden of large particulates and most intact cells while preserving EVs and viruses in the filtrate, seawater was sequentially passed through 0.8 µm and then 0.45 µm filtration. Briefly, the 100 L depth sample was first filtered through a 0.8 µm membrane into a clean reservoir. The resulting filtrate was then immediately filtered through a 0.45 µm membrane, producing the operational <0.45 µm fraction for downstream concentration. Throughout filtration, care was taken to maintain low shear and operate within manufacturer-recommended flow and pressure ranges to avoid unnecessary stress on vesicles and viral particles. Filtrates and reservoirs were kept cold during handling. The outcome of this stage was a <0.45 µm feed stream for tangential flow filtration (TFF), with the volume remaining approximately 100 L aside from minor handling losses.

Tangential flow filtration (TFF) for concentration and buffer exchange

The <0.45 µm fraction was concentrated using tangential flow filtration (TFF) equipped with a 100 kDa MWCO cartridge. The filtrate was recirculated per the instrument and cartridge guidance until the sample volume was reduced from ~100 L to approximately 50–100 mL of retentate. In addition to concentrating particles, the TFF step was used to transition the sample into a buffer compatible with downstream EV column clean-up. Buffer exchange was performed by adding multiple retentate-equivalent volumes of the chosen EV/virus-compatible buffer (e.g., filtered seawater or PBS) to the retentate and reconcentrating, repeating as needed in line with manufacturer recommendations. During TFF, the retentate was maintained cold, and operating conditions were managed to avoid excessive transmembrane pressure, foaming, or vigorous agitation. This stage produced a 50–100 mL TFF concentrate enriched for EVs and viruses, compatible with the column.

EV column clean-up and elution to a final 1 mL product

To further remove soluble components and improve sample cleanliness for downstream analyses, the TFF concentrate was polished using commercial EV columns (Takara or Qiagen, depending on availability and cruise workflow). Columns were equilibrated following the manufacturer’s kit instructions, including the specified buffers, volumes, and centrifugation/flow conditions. The TFF concentrate was then loaded onto the column(s); when necessary, multiple columns were used to stay within the recommended sample loading limits. After loading, columns were washed with the supplied wash buffer to reduce carryover of non-target material. Vesicle/virus-enriched fractions were then eluted to a final total volume of 1 mL per depth, either as a single elution or by pooling elutions as appropriate for the kit format and loading strategy. The result of this step was a clean, small-volume EV/virus-enriched eluate suitable for archiving and analytical workflows.

Aliquoting, cryopreservation, and storage

Immediately after elution, the 1 mL product was gently mixed (avoiding vigorous vortexing) and distributed into 10 low-binding tubes at 100 µL per tube. Aliquots were snap-frozen in liquid nitrogen and then transferred to −80 °C for long-term storage. Each tube was labeled with the relevant collection and processing identifiers, including cruise ID, station, cast, depth, date, the designation “EV/virus column eluate,” and the aliquot number (e.g., 1/10–10/10), ensuring clear linkage between the archived material and the associated sampling metadata.