The brain, often considered the central command center of the human body and the uppermost pillar of the central nervous system (CNS), is a remarkable and intricate organ. Safeguarded by the skull, the brain’s well-being is further protected by a formidable barrier called the blood-brain barrier (BBB). This remarkable barrier serves as a sentinel, meticulously regulating the passage of molecules between the bloodstream and the brain parenchyma to maintain the delicate balance of the neural microenvironment (Bang, Jeong, Choi, & Kim, 2019).

The BBB is defined by four essential characteristics (Fig. 1) (Li et al., 2020):

Cell composition variety: The BBB comprises three primary cell types: brain microvascular endothelial cells (BMECs), perivascular astrocytes, and pericytes. These cells collaborate to form a complex system designed to protect the brain.

The role of tight junction proteins: Tight junction proteins are instrumental in constructing the primary structure of the BBB. These proteins establish a barrier that is tightly sealed and impervious to most substances. This network of proteins is essential for maintaining the barrier’s integrity.

Selective permeability: The BBB is highly selective in its permeability. While essential molecules like water, gases, and lipid-soluble substances can easily pass through cell membranes, others, such as glucose and insulin, require specialized transport proteins. In contrast, some substances are actively regulated and selectively transported to prevent their entry into the brain.

Influence of fluidic flow and shear stress: Fluidic flow, driven by cerebrospinal fluid and blood circulation, generates shear stress around BBB cells. This mechanical force, especially pronounced for endothelial cells (BMECs), plays a crucial role in cell differentiation and the formation of tight junction proteins. It underscores the dynamic nature of the BBB.

The concerted action of these features ensures the BBB’s efficacy in preserving the sanctity of the brain’s microenvironment. Several diseases have been unequivocally linked to compromised BBB function. Additionally, the tight junctions within the BBB can hinder the penetration of therapeutic drugs into the CNS. For example, Alzheimer’s disease (AD), a neurodegenerative condition characterized by neuronal cell death, neuroinflammation, and the accumulation of neurotoxic protein plaques, has been extensively studied through both postmortem human research and animal models (Franco & Cedazo-Minguez, 2014).

The BBB allows the passage of nutrients and regulates metabolite influx through passive and active mechanisms (Fig. 2). The endothelial cell (BMEC) membrane’s lipid composition allows transient gaps, permitting the passage of small molecules or trace amounts of water (Pifferi, Laurent, & Plourde, 2021). Embedded proteins, including transporters, mediate molecule transport (Peetla, Stine, & Labhasetwar, 2009). Small molecules (<500 Da) can freely diffuse through the BBB via the paracellular route, while larger molecules are transported through carrier-mediated, receptor-mediated, or absorption-mediated transport (the transcellular route) (Pardridge, 2005, Preston et al., 2014, Tran et al., 2022). Various transporters and carriers, such as amino acid transporters, carbohydrate transporters, fatty acid transporters, and organic anion/cation transporters, facilitate the influx of nutrients. Receptor-mediated transcytosis involves macromolecules binding to receptors, while absorption-mediated transcytosis relies on positively charged carriers. Efflux transporters, including ATP-binding cassette (ABC) transporters and the Aβ clearance system, prevent metabolites from entering the brain and facilitate the removal of endogenous metabolites from the CNS (Preston et al., 2014).

Therapeutic drugs for brain diseases should penetrate the BBB, which must be evaluated with in vitro models. Traditional in vitro drug screening primarily relies on monolayer or 2D cell cultures using various cell types to assess drug absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox). However, these models often fail to replicate the realistic microenvironment and cell-to-cell interactions. Furthermore, animal models, although valuable in representing complex biological systems, are fraught with limitations, including issues related to detection methods, time, cost, and ethical considerations. Notably, results obtained from animal studies frequently diverge from human responses due to species-specific variations in physiology (Bang et al., 2019). Consequently, developing reliable and biomimetic in vitro BBB research models has emerged as a burgeoning field of study in recent years (Fig. 3).