What is a Vesicular Transport Assay

Why inside-out?

ABC efflux transporters in living cells have their substrate-binding site facing the cytoplasm — inaccessible from outside the cell. To study the transporter directly, the membrane must be inverted. In inside-out vesicles, the binding site faces the incubation buffer, allowing the test compound to interact with the transporter without first crossing a cell membrane. This eliminates passive permeability as a confounding variable — the major limitation of cell-based transport assays for lipophilic compounds.

Vesicular transport vs cell-based assay

Cell-based bidirectional transport assays (Caco-2, MDCKII-MDR1) measure the efflux ratio of a compound across a cell monolayer. For high-permeability compounds, passive diffusion dominates and the efflux ratio collapses toward 1.0 — masking active transporter efflux. The vesicular transport assay has no such limitation because no membrane crossing is required to reach the transporter binding site.

Ready-to-use vesicle kits

Cell4Pharma ABC Transporter Vesicle Kits (distributed by SeamlessBio in DACH) provide pre-prepared, QC-validated inside-out membrane vesicles for in-house DMPK transporter testing — covering BSEP, P-gp, BCRP, MRP1–MRP5, MRP8, and Control vesicles. 100 reactions per kit, stored at −80°C.

Key Facts

• Inside-out vesicles are prepared from cells (Sf9 insect cells or HEK293) overexpressing a specific ABC transporter — the membrane inverts during preparation, exposing the substrate-binding site outward
• ATP-dependent substrate accumulation inside vesicles = specific transporter activity; AMP control = non-specific background
• Does not require cell culture at time of assay — vesicles are stored at −80°C and thawed on demand
• Passive permeability of the test compound does not affect the assay — a key advantage over cell-based assays for high-permeability compounds
• The only viable method for BSEP, MRP2, MRP3, MRP4, MRP5, MRP8 — no standard cell monolayer model exists for these transporters
• Results available same day — typical incubation 5–30 minutes per transporter
• Accepted by FDA, EMA, ICH M12 for DDI inhibitor IC50 determination

Further Reading

• Inside-Out Vesicles Explained — Complete Guide
• Vesicular vs Cell-Based vs ATPase Assay
• What is BSEP?
• What is P-glycoprotein (P-gp)?

SeamlessBio GmbH · Passau, Germany · info@seamlessbio.de · +49 851 37932226