Recent advances in stem cell technology, tissue engineering, and microfluidics have led to rapid advances in the complexity of in vitro models of the blood–brain barrier (BBB). Stem cell technology provides a reliable source of human, brain-specific cells: iPSC-derived human brain microvascular endothelial cells (dhBMECs) exhibit many of the hallmarks of human BMECs [
1‐
29], a long-standing problem in developing BBB models [
30‐
32]. Additionally, protocols for iPSC-derived astrocytes, pericytes, microglia and neurons have been developed to facilitate modeling of the neurovascular unit [
33]. Similarly, advances in tissue engineering and microfluidics provide the tools for organization of perfusable microvessels or microvascular networks [
34,
35]. Diverse BBB-on-a-chip models have emerged over the last 5 years, they can generally be classified as: (1) two-dimensional microfluidic models, (2) hybrid microfluidic models, (3) three-dimensional templated models or (4) self-organization models. Two-dimensional microfluidic models incorporating a permeable membrane (resembling that of a traditional Transwell
® assay) are extremely valuable for applications such as drug screening or measures of electrical resistance, however, these models do not recapitulate many aspects of physiological BBB function [
36‐
39]. Hybrid microfluidic models capture more complexity but lack homogenous cell-ECM interactions and cylindrical geometry and are thus not able to respond to vasodilation/constriction [
40,
41]. Templating approaches support generation of singular cylindrical microvessels embedded within an extracellular matrix that can be integrated into a flow system for live-cell imaging [
23,
24,
42‐
44]. Lastly, self-organization approaches that mimic vasculogenesis and/or angiogenesis have emerged to generate multicellular models of brain microvascular networks [
45,
46]. Since animal models do not always recapitulate human physiology or disease [
47,
48], in vitro models can provide an important link between human physiology and animal models.
The value of BBB models in basic and translational research is dependent on the ability to recapitulate in vivo and ex vivo studies. The fidelity of the model is usually dictated by the purpose and the processes under study. More reductive models will naturally recapitulate fewer characteristics of the BBB, while more complex models attempt to recapitulate more characteristics but are usually lower throughput. In all cases, benchmarking to in vivo studies is key to establishing physiological relevance.
Benchmarking is surprisingly challenging, in large part because much of our knowledge about BBB structure and function is derived from in vitro studies. Here we describe 12 design criteria for tissue engineering the human BBB. This is not intended to be a complete checklist of benchmarks for model validation, but a limited set of parameters associated with structure (ultrastructure, wall shear stress, geometry), microenvironment (basement membrane and extracellular matrix), barrier function [transendothelial electrical resistance (TEER), permeability, efflux transport], cell function (expression of BBB markers, turnover), and co-culture with other cell types (astrocytes and pericytes). Depending on the purpose of the model, the specific benchmarks may vary and may not include all those listed here. Wherever possible, benchmarks are suggested based on imaging or direct measurement in humans or animal models.