Table 1.

Biosafety recommendations for work involving the isolation and use of extracellular vesicles (EVs) from SARS-CoV-2 infected samples.

Training	Advanced biosafety training must be provided to laboratory personnel conducting research with SARS-CoV-2 infectious material. Seek support from the institutional office of environmental health and safety (EHS).
Personal Protective Equipment (PPE)	Recommended use of rear-opening lab coat, double gloves, face mask, and goggles. Face shield may also be advisable. Reusable PPE should be decontaminated or autoclaved (e.g., clothes or linens prior to laundering).
Aerosol Containment	Manipulation of SARS-CoV-2 infected materials should be conducted inside a Class II (or better) biological safety cabinet (BSC). Avoid the generation of aerosols outside the BSC.
Centrifugation	Should be conducted using centrifuge safety buckets or sealed centrifuge tubes in sealed rotors.
Nanoparticle Tracking Analysis	To avoid the generation of aerosols with contaminating live viral particles (e.g., during elimination of air bubbles from the loading syringe before running samples through the NanoSight), it is recommended to use inactivated EVs to measure the size and concentration of the particles.
Decontamination	Contaminated laboratory waste and equipment should be decontaminated, e.g., autoclaved or decontaminated with fresh solution of 10% chlorine bleach (final concentration against the volume of waste) before disposal or washing. Liquid waste decontaminated with 10% chlorine bleach should be allowed at least 30 minutes of contact time. If using 10% bleach solution on work surfaces and equipment, allow it to air dry then wipe with 70% ethanol to prevent rusting of stainless steel surfaces.
Virus inactivation (49)	To minimize exposure risk, virus inactivation in samples/aliquots not needed for live EV experiments is recommended as soon as preparations are generated: For nucleic acid assays: lyse EV preparations in TRIzol (at least at a 10% final concentration and with 10 minutes incubation at room temperature), and freeze at -80 °C until use. For immunohistochemistry and microscopy: fixed samples in paraformaldehyde (e.g., at least at 1% final formaldehyde concentration with 1 hour or longer incubation at room temperature). For biochemical assay development: incubate with 0.5% beta-propiolactone for 16 hours at 4 °C followed by 2 hours incubation at 37 °C), or apply heat for 5 minutes at 100 °C or 45 minutes at 56 °C).
Laboratory accidents	Should be immediately reported to the Principal Investigator and EHS. Maintain a biological spill kit in the laboratory to facilitate spill response and decontamination.
Airflow	In addition to the use of biosafety cabinet, inward directional room airflow is advisable.
Immunizations	Vaccines should be obtained for those engaged in research with potential exposure to the specific infectious agent.

For a comprehensive review of advanced biosafety laboratory procedures, we recommend the Biological Safety BSL3 Laboratory Manual from Yale University (https://ehs.yale.edu/sites/default/files/files/bsl3-lab-manual.pdf).