Additional Table 2.

Technical strategies applied to the establishment of in vitro neurovascular unit (NVU) models

Model	Materials of membrane	Cell types	Advantages	Limited	References
Transwell static	Poly tetra fluoroethylene, Porous polycarbonate (PC)	Rat brain endothelial cells GP8, RBE4, hCMEC/D3	Easy to set up, moderately scalable, low-cost	Unable to simulate NVU dynamic model, lack of physiological shear stress to limit endothelial cell blood-brain barrier (BBB) phenotypic differentiation, relatively low endothelial resistance, and high permeability of hydrophilic substances	Bian et al., 2019;
					Li et al., 2019;
					Brown et al., 2020; Rumianek and Greaves, 2020
Dynamic microfluidic device	PC	B.end3 endothelial cells or co-culture with astrocytes	Real-time visualization BBB and transmembrane endothelial resistance (TEER)	Weak ability to regulate microenvironment	Booth and Kim, 2014
Dynamic synthetic microvascular device	Polydimethylsiloxane (PDMS)	RBE4 (intra luminal), astrocytes (extra luminal)	Low cost, simulated microcirculation environment, physiological fluid flow, shear stress, allow long-term cell culture, real-time optical monitoring	High throughput drug screening and TEER real-time detection are not available	Prabhakarpandian et al., 2013
Dynamic NVU chip device	PC	RBE4, neurons, astrocytes and microglia	Low cost, realize NVU interaction	The apparatus is complicated and it is difficult to culture cells for a long time	Achyuta et al., 2013
Dynamic New baby BBB chip device	PDMS	Rat brain endothelial cells, astrocytes	Simulation of shear flow in vivo environment, real-time visualization, direct measurement of dynamic process	Technology is not mature enough	Deosarkar et al., 2015
Microfluidic-on-chip	PDMS chip, porous polycarbonate film	Human brain neurons, astrocytes, pericytes, and BMECs	Low reagent consumption, high throughput, easy integration. Controllable physical parameters, accurate simulation of central nervous system environment. Low cost fabrication, flexibility in the design, visualization of cells is possible, consider the effect of sheer stress. Immediate permeability measurements. Improvement in paracellular barrier functions	Highly specialized equipment, limited materials and expensive reagent consumption make it impossible to quantify cavity membrane shear stress and transendothelial resistance, lack of high-throughput, complex process technically, not ideal linear kinetic	Sankar et al., 2017; Mittal et al., 2019; Bhalerao et al., 2020; Guo et al., 2021; Staicu et al., 2021
Three-dimensional printing	Gelatin/alginate hydrogel, swarm hydrogel, collagen, gelatin, fibrin, gellan gum, hyaluronan, self-assembling peptide, elastinlike polypeptide, polyethylene glycol	Neuroblastoma cell line (SH-SY5Y), induced pluripotent stem cells, and neural stem cells, Schwann cells	Quickly, high cost performance, complex geometric features, low cost, multi-material and multi-function, elastic, high resolution	High cell survival rate cannot be guaranteed. Bio-inks have a limited shelf life. A variety of cells in the system may cause an immune response. The mechanical strength of tissue scaffolds is low	Potjewyd et al., 2018; Fantini et al., 2019; Gong et al., 2020
In-Silico			Short time, low cost, low risk. Real-time observation of the interaction between NVU cells. Quantized molecular signal	In vivo or in vitro verify are needed	Katara, 2014; Li et al., 2020a