Extracellular vesicle experiments are highly sensitive to cell state, conditioning strategy, isolation workflow, normalization and analytical bias. EV-iTEC therefore provides quality control, experimental design and reporting tools to support transparent, reproducible and method-aware EV workflows, featuring MiTEC (Minimum Information for Transparent EV Characterization).

Before starting an EV experiment, ask yourself:

What is the biological question?
What result would contradict my hypothesis?
Which control would convince a sceptical reviewer?
Why was this isolation strategy chosen?
How will the data be normalized?
Which information must be reported?

EV-iTEC tools support experimental planning, QC and reporting. They do not replace scientific judgement, experimental controls or critical interpretation.

MiTEC was developed by the EV-iTEC Core Facility to support practical experimental planning and structured documentation of extracellular vesicle studies in alignment with MISEV2023 principles. MiTEC is not intended to replace either MISEV or EV-TRACK. While EV-TRACK serves as a community-driven platform for transparent reporting and centralized documentation of completed studies, MiTEC focuses on the earlier stages of experimental design, feasibility assessment, pilot planning, and routine reporting. The two approaches are complementary and together contribute to improved transparency, reproducibility and reporting quality in extracellular vesicle research.

EV-iTEC · MiTEC v2

MiTEC Experimental Design Assistant

MiTEC — Minimum Information for Transparent EV Characterization. Three-stage workflow before any EV isolation: (1) pre-experiment feasibility check with scored risk assessment, (2) interactive pilot checklist as scale-up gate, (3) full MiTEC documentation form with exportable report. No EV interpretation without cell-state documentation.

Experiment input

Complete before scheduling any EV isolation. Goal: expose avoidable design risks early, not to block experiments.

Biological system

Cell type / model cell line, primary cells, organoids

Expansion medium routine culture before EV collection

EV collection medium after switch at sufficient confluence

Mycoplasma status

Passage number

Passage range defined?

Treatment / stimulus or "none"

Cell state at harvest

Viability at harvest (%)

Confluence before medium switch (%) EV-iTEC minimum: ≥ 75%

Confluence / density at harvest (%)

Cell number at harvest documented?

Conditioning time after medium switch (h) EV-iTEC maximum: 48 h

Hypoxia during conditioning?

EV experiment design

Planned conditioned medium volume (mL)

Biological replicates (n)

Prior pilot performed?

Isolation method

Normalization strategy

Readouts planned

Particle measurement

Protein / contamination readout

Marker / phenotyping readout

Notes / special conditions

Design score

–

QC-oriented, not regulatory

Recommendation

–

proceed / revise / pilot first

Scale-up risk

–

especially relevant for ≥ 500 mL

Interpretation

Fill in the left panel to see the assessment.

Risk indicators

Cell-state risk

–

Scale-up risk

–

Reporting risk

–

Required before EV interpretation

Method-specific note

Required pilot items

All 5 must be completed before any scale-up > 200 mL is approved. No large-scale UC or DG without documented pilot QC.

Small-scale conditioned medium collected (10–50 mL) Same cell system, same medium composition, same conditioning time as planned scale-up

Cell count and viability documented at harvest Hemocytometer or automated counter; > 85% viability required to proceed

NTA or NanoFCM size/concentration check performed Mode diameter, concentration (particles/mL) and size distribution recorded

Protein measurement done (microBCA or BCA) EV pellet or fraction; particle-to-protein ratio (PPR) calculated and recorded

Isolation method documented (rotor, RCF, k-factor, time, temperature) Required for reproducibility; enter full parameters in MiTEC form (Tab 3)

Recommended pilot items

Strongly encouraged for new cell systems, new methods, or scale-up > 500 mL.

Western blot: positive markers (CD63, CD9, TSG101) Confirms EV enrichment

Lipid measurement (SPV assay) for lipid/protein or lipid/particle normalization Useful when absolute cell number normalization is not straightforward

Density gradient fraction validation (peak fraction identity confirmed) For UC + DG: confirm fraction density and marker distribution before scale-up

Pilot score

0 / 5

Complete required items to see scale-up recommendation.

Pilot QC metrics

Record results from the pilot run. Values are included in the MiTEC summary.

Particles / mL (NTA / NanoFCM)

Mode diameter (nm)

Protein (µg/mL or total µg)

PPR (particles / µg protein)

Pilot notes / observations

EV-iTEC scale-up rules

Condition	Requirement
< 200 mL	Pilot encouraged, not mandatory
200–500 mL	All 5 required pilot items
> 500 mL	All 5 required + ≥ 2 recommended items
UC + DG, any volume	All 5 required + WB marker check
New cell line / new medium	Full pilot regardless of volume

EV Biological Use Verification

For any experiment in which EVs are used as biological agents — e.g. uptake, targeting, ligand binding, protein corona studies, biodistribution, animal experiments or engineered EV approaches — EV identity and integrity should be verified at the time of biological use and not only after isolation.

Downstream biological use planned?

Type of biological use

Verification notes

Minimum documentation before biological interpretation

Particle concentration and size distribution NTA, NanoFCM, TRPS or equivalent, ideally checked after storage, labeling, binding or concentration steps.

Particle-to-protein ratio or equivalent purity metric Documents whether protein co-isolates, aggregation or particle loss may confound the biological readout.

Retention of at least one EV-associated marker Confirms that the preparation used in the assay still represents the characterized EV population.

Retention of the experimentally relevant marker, ligand, antibody or engineered surface feature Particle count can remain stable while the biologically relevant EV surface feature is lost, masked or altered.

Appropriate assay-matched controls included Examples: mock isolation, buffer control, detergent-disrupted EVs, protein/ligand-only control or EV-depleted fraction.

0 / 5

Not applicable.

Set "Downstream biological use planned?" to Yes to activate this verification gate.

Interpretation note

Biological effects should not be attributed to EVs if EV identity, integrity or the experimental surface modification were not verified at the time of use.

MiTEC reporting form

Document all parameters before interpreting EV results. Missing values stay visible as "not determined" — never silently omit. Compatible with MISEV2023.

01 · Cell system

Cell type / line *

Species / origin

Passage number *

Mycoplasma status *

Seeding density (cells/cm²)

Confluence at medium switch (%) *

Harvest confluence / density (%) *

Cell number at harvest (× 10⁶) *

Viability at harvest (%) *

Viability method

02 · Culture & conditioning

Expansion medium *

EV collection medium *

Serum condition *

Conditioning time (h) *

Culture volume (mL) *

Vessel / format

Hypoxia condition

Treatment / stimulus *

03 · Isolation

Isolation method *

Pre-clearance steps

Rotor / k-factor

RCF (× g)

UC time (min)

Temperature (°C)

Density gradient composition

Fraction collection scheme

04 · Quantification & QC

Particle concentration (particles/mL)

Mode diameter (nm)

Measurement instrument

Protein concentration (µg/mL)

Protein assay

PPR (particles / µg protein)

Normalization strategy *

Marker / QC readouts

05 · Study design & reporting

Biological replicates (n) *

Technical replicates (n)

Controls

Notes / deviations from SOP

Copyable MiTEC summary

Auto-generated from Design check (Tab 1) and MiTEC form (Tab 3). Use for sample submissions, lab notebooks or pre-analysis notes. Missing fields remain visible.

Summary status

Not yet generated.

Click "Update summary" or navigate to this tab to auto-generate from your inputs.

MiTEC minimal reporting checklist

Category	Minimum required
Cell model	Cell type, passage, expansion + collection medium, treatment, hypoxia, conditioning time
Cell state	Confluence at switch and harvest, viable cell number, viability, mycoplasma status
Replicates	Biological n (≥3), technical replicates, batch / date structure
Isolation	Input volume, method, rotor / RCF / k-factor / time or column equivalent
QC	Particles, protein, PPR, marker readouts, negative / blank controls
Normalization	Per viable cell number, protein, or multi-parameter normalization

Important note: MiTEC is a practical EV-iTEC design and reporting aid. It is not a replacement for project-specific experimental planning, MISEV2023-based reporting, statistical consulting or biological validation. The assistant intentionally flags missing cell-state information because EV interpretation without it is weak.