Table 1.

Blood–brain barrier validation markers.

Category	Property	Relevance	Validation	Key references
Validation of cell lineage	Monolayer of thin cells with large surface area	All studies	Visualization, F-actin staining	4,5
	Expression of endothelial markers		Von Willebrand’s Factor/PECAM-1	6–8
Tight junctions	Occludin claudin-5 ZO-1	Studies of tight junctions – transendothelial transport and uptake studies – Cell polarization	mRNA and protein expression – localization	9–11
	High junctional tightness		TEER and permeability measurements	12–16
Efflux transporters	P-pg	Transendothelial transport and uptake studies – drug delivery to/through the BBB – toxicity	mRNA and protein expression – Cellular uptake or efflux in absence/presence of inhibitors – bi-directional transport studies	17,18
	BCRP			19–22
	Mrp			23–25
SLC expression	Glut-1	Transendothelial transport and uptake studies – drug delivery to/through the BBB. Brain nutrition studies	mRNA and protein expression – Cellular uptake in absence/presence of inhibitors – transendothelial transport studies	26–28
	LAT-1			29,30
	MCT-1			31–33
Receptor systems	Transferrin receptor	Studies of receptor-mediated transport, brain nutrition studies	mRNA and protein expression – transferrin uptake – transendothelial transport of iron	34–36
Responsiveness to regulation from NVU cells	Induction by astrocytes	Studies of cell regulation and NVU signalling	Regulation of TEER, P-gp expression and cell morphology	37–40
	Induction by pericytes		Regulation of TEER, proteins involved in vesicular transport	41,42