Evaluating sex as a biological variable in in vitro blood–brain barrier models: insights from primary mouse brain endothelial cells

Preclinical neurological research relies predominantly on male animals, despite well-documented sex differences in neurological diseases, which ultimately may result in sex-dependent treatment efficiency. A key player in neurological disease treatment is the blood-brain barrier (BBB), the barrier property of brain capillaries, which tightly regulates molecular exchange between the blood and the brain. The BBB represents a major obstacle to brain drug delivery due to its tightness and presence of drug efflux pumps, with some studies suggesting that the BBB properties may differ between sexes. However, in vivo evidence is limited, and whether primary in vitro BBB models, commonly used to evaluate the permeability of novel drug candidates, display sex-dependent differences, lacks attention. With this study, we therefore aimed to investigate if a mouse in vitro model of the BBB displayed sex-dependent differences in BBB morphology and phenotype, and therefore whether sex should be considered a critical variable in its use. Primary mouse brain endothelial cells (PMBEC) were isolated from cortices of sexually mature C57Bl/6 mice. Transendothelial electrical resistance (TEER) measurements and transport of the paracellular marker [14C]-mannitol were used to evaluate monolayer tightness. Gene and protein expression of tight junction proteins, selected transporters and receptors as well as efflux transporters were assessed. P-glycoprotein (P-gp) function was evaluated in bidirectional [3H]-digoxin transport studies. Female- and male-derived PMBECs grew in monolayers, expressed the endothelial marker von Willebrand factor and showed elongated spindle-shaped morphology typical of endothelial cells. Female- and male derived PMBEC monolayers exhibited comparable barrier properties as reflected by TEER measurements and mannitol permeability. Tight junction mRNA and protein expression did not differ between sexes and displayed similar expression levels of key transporters. Lastly, P-gp and breast cancer resistance protein were detected in PMBECs of both female and male origin and P-gp function was similar in the two sexes. Our present study shows that PMBECs do not differ substantially between sexes. However, as this is the first study of its kind, it warrants further investigations into sex differences in PMBECs and whether these fully translates to the in vivo BBB.