Establishment of an in vitro blood-brain barrier model by cocultuting brain microvascular endothelial cells and pericytes

Abstract

Objective To establish a stable in vitro model-of blood-brain barrier （BBB） simulating in vivo state using the primary-cultured rat brain microvascular endothelial cells （BMVECs） and pericytes. Methods The primary rat BMVECs and pericytes were isolated, purified and cultured. The isolated cells were identified by immtmocytocliemical staining method. An in vitro model of BBB was construeted using Transwell inserts （pore size 0.4 μm） coculture. Its barrier function was evaluated by the 4-hour leakage test, tight junction protein identification, transendothelial resistance detection, and permeability test. The difference between the cocultured model and simple BMVEC model across the membrane resistance values, and the permeability difference of the small molecule sodium fluorescein （Na-F） were compared. Results Confluent BMVEC monolayers demonstrated a typical cobblestone appearance and the peficytes displayed irregular shape and overlapping growth. Immu- nodouble labeling technique identification showed that the pericytes positively expressed α-smooth muscle actin （α-SMA） and neuron-glial antigen 2 （NG2）; after the fusion of cocultured model endothelial cells, the surface leakage test became positive; immunocytochemical staining shows that a continuous and dense tight junction formed between the endothelial cells; compared to the BMVEC model, the transendothelial electrical resistance of the cocultured model increased significantly （190. 762± 10. 326 Ω/cm^2 vs. 96. 503 ±8. 012 Ω/cm^2; t =-24. 489, P 〈0. 01）, and the permeability decreased significantly （56. 149% ± 3. 572% of the single endothelial model; t = 19. 330, P 〈 0. 01）. Conclusions The primary isolated rat BMVECs and pericytes cocultured the morphology, structure and barrier function of in vitro model have the basic characteristics of BBB, and they have provided a useful tool for the research of BBB. Key words: Blood-Brain Barrier; Pericytes; Endothelial Cells; Cells, Cultured; Rats