T175 flask

The cells were seeded into T175 flasks (Thermo Fisher Scientific). At 80% confluence, the cells were washed three times with PBS (Gibco by Life Technologies), the medium was changed to serum‐free medium, and the cells were further incubated for 24 h. SW480_EV‐S and SW480_EV‐M were isolated from the conditioned media by sequential centrifugation and ultrafiltration. Briefly, cell debris and floating cells were removed by centrifugation at 300 g for 10 min and 4500 g for 5 min, and the supernatant was stored at −80 °C. The supernatant was thawed at 37 °C, and medium EVs were isolated by centrifugation at 17 000 g (11 930 r.p.m., k‐factor 2112) for 30 min using a fixed‐angle Sorvall SS‐34 rotor (Kendro Laboratory Products, Newtown, CT, USA). The pellet was resolved in serum‐free medium and concentrated using Amicon Ultra‐4100 kDa Centrifugal Filter Devices (Merck Millipore, Cork, Ireland). Prior to small EV isolation, to exclude particles larger than 220 nm, the 17 000 g supernatant was gently filtered through 0.22‐μm filter (Merk Millipore). Small EVs were isolated by Centricon‐70 Plus 100 kDa Centrifugal Filter Columns (Merk Millipore) and centrifuged at 3500 g for 15 min with a washing step in PBS at 3500 g for 10 min. All centrifugations were performed at room temperature. All samples were stored at −80 °C.

To determine the morphological maintenance of BDNF-eMSCs compared to the naïve MSCs, 0.6 × 10⁶ cells of BDNF-eMSCs and 1 × 10⁶ cells of naïve MSCs, on average, were seeded into T175 flasks (Thermo Fisher Scientific, Waltham, MA, USA). After 3–4 days, cell morphology was observed by optical microscope (NIKON, Tokyo, Japan), and images were captured (original magnification 100×).

Acanthamoeba castellanii cells (ATCC 30010) cultivated in PYG (Peptone, Yeast extract, Glucose) medium were used to produce Tupanvirus Deep Ocean and Tupanvirus Soda Lake. Suspensions containing 7×10⁶ cells plated in T175 flasks (Thermo Fisher Scientific, USA) were inoculated with each virus at an MOI of 0.02 and incubated at 30 °C. After complete lysis of the cells, each virus supernatant was collected and centrifuged at 1000g. The obtained supernatant was then filtered through a 0.8-μm membrane to remove amoeba debris. Each viral pellet was submitted to three cycles of wash with Page’s modified Neff’s amoeba saline (PAS) by ultracentrifugation at 14,000g for 1 h. Finally, each virus was purified through ultracentrifugation across a 25% sucrose cushion at 14,000g for 1 h.