Table 2.

Recommended reporting guidelines for EV loading studies. Guidelines are intended for use in addition to existing MISEV criteria on EV production, isolation/separation, and characterisation.

Experimental Parameter	Required Information
EV Isolation	Details of the isolation method applied (e.g. ultracentrifugation, ultrafiltration, SEC) with detailed protocols (including centrifuge model and rotor type, speed and time of centrifugation, brand and pore size of filters, number of centrifugation cycles or filtrations, and storage conditions following isolation, where applicable).
	Where possible, authors should demonstrate that the EV isolation strategy has not caused morphological changes, such as through the application of high g-forces during ultracentrifugation.
EV Characterisation	For accurate reporting of EV numbers used in loading studies, EV quantification should ideally be estimated by measurement of particle number, for instance by nanoparticle tracking analysis or nano/high resolution flow cytometry. If protein amount is to be used to quantify EVs, authors should make clear the limitations of quantification by protein content. Also, enrichment of marker proteins should be demonstrated when compared with cell lysates to assess purity.
EV Loading	EV/sample mixtures should be precisely reported. Information should include: the amount of molecular cargo, the number of particles/EVs, and the loading solvent (PBS, electroporation butter, etc).
	Full details of loading protocols should be provided: Incubation - temperature and time. Sonication - sonic bath/probe model, sonication settings, sonication time and the number of cycles. Detergent permeabilisation - type of detergent, concentration, temperature and incubation time. Electroporation – type/model of electroporator used, full details of electroporation parameters (e.g. voltage, pulse width, pulse number), and the electroporation buffer used.
	The method applied for the subsequent isolation of loaded EVs should be detailed (ultracentrifugation, ultrafiltration, SEC), including centrifuge model and rotor type, speed and time of centrifugation, the number of wash/filtration steps, and the type of filter used (where relevant).
	Where possible, EV quantification should be performed following isolation of loaded EVs to establish EV loss following ultracentrifugation, filtration, etc.
	Authors should demonstrate, where possible, that EV loading has not caused morphological changes to EVs (e.g. via transmission electron microscopy) or altered the biological properties of the particles (e.g. cellular targeting, uptake, biodistribution).
	Authors should state whether loaded EVs were used immediately for further analysis or stored for subsequent analysis. If the latter, storage conditions and time should be specified.
Loading Assessment	Full details of analytical technique used for detection of molecular cargo to assess EV loading should be detailed including, where applicable, instrument type and model, detection parameters, any sample treatment required for analysis.
	Clarify whether loading efficiency was determined via measurement of unloaded cargo (e.g. remaining in supernatant following loaded EV isolation) or via measurement of loaded EVs (e.g. following lysis of loaded EVs to release cargo). One should also discriminate luminal loading from simple association through incubation with relevant enzymes (e.g. RNase) or separation using density gradient UC.
	If applicable, authors should state the protocol used to lyse loaded EVs and, if possible, should demonstrate that any EV lysis procedures (e.g. detergent addition) do not interfere with detection of loaded molecular cargo.
	The success of the loading procedure should be reported in the form of loading efficiency or encapsulation efficiency, including specification of the number of sample replicates and the reproducibility of the loading protocol.
Functional Delivery	When loaded EVs are to be applied in in vitro or in vivo studies, appropriate controls should be conducted. This should include EV only, cargo only, and negative controls, all of which should be subjected to the same treatment as the loaded EVs.
	Authors should demonstrate dose-dependent effects of loaded EVs on an in vitro or in vivo response.