Co-Culture and Multicellular Models of the Blood-Brain Barrier for Drug Testing

The blood-brain barrier (BBB) is made up of endothelial cells that line the capillaries in the brain. The endothelial cells are joined by tight junctions that prevent most molecules from passing freely through. This makes the BBB a formidable physiological barrier to the movement of molecules. The properties of the BBB have made the delivery of drugs to the central nervous system (CNS) a formidable challenge. A large proportion of potential CNS drugs do not cross the BBB to reach their targets. In vitro BBB models allow scientists to study and screen compounds under controlled conditions.

Creative Biolabs provides BBB research platforms that include co-culture and multicellular BBB models that will help researchers to better understand and assess drug transport and permeability through the BBB in vitro.

Cellular Composition and Functions of BBB

The BBB is a selective semipermeable membrane formed by brain capillary endothelial cells, astrocytes, and pericytes. The BBB selectively excludes harmful blood substances from entering the brain while ensuring the rapid transport of nutrients and metabolic products. Table 1 shows the cellular composition and functions of the BBB.

Table 1 Cellular composition of BBB.

Single-Cell Culture Models of the Blood-Brain Barrier

Traditional single-cell culture models are generally based on the culture of brain microvascular endothelial cells. Although this cell model can partially mimic some properties of endothelial cells, it has the following shortcomings in simulating the BBB microenvironment.

• The single-cell culture model is in isolation from other cell types, such as astrocytes and pericytes, and the lack of their support and interactions cannot truly reflect the physiological and functional status of the BBB in vivo, so it has low predictive accuracy in drug tests.
• Moreover, most traditional models are static systems that do not consider the fluid shear stress and dynamic blood flow environment in vivo, which limits their application in simulating complex neuropathological processes such as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS).

The use of traditional single-cell BBB models is often limited by its inability to simulate in vivo environment, cell-cell interactions, and dynamic physiological states. Therefore, more complex co-culture models and three-dimensional dynamic systems need to be established.

Co-Culture Models of the BBB: Endothelial Cells and Astrocytes

Co-culture models involve co-culturing endothelial cells with astrocytes to allow for cell-cell interactions. Astrocytes can significantly enhance the BBB properties of endothelial cells in co-culture systems.

This can be performed in a variety of culture methods. Three common co-culture methods include Transwell system, direct contact culture, and three-dimensional culture.

Table 2 Co-culture models and their advantages and limitations.

The Transwell system is well suited to initial screening and functional studies, direct contact culture provides a better environment for cell-cell interactions, and 3D culture more closely mimics the complexity and dynamic in vivo BBB environment, and thus is an important area for future research.

Figure 1 The establishment of a co-culture in vitro BBB model.^1,2

Retinoic Acid-Enhanced Multicellular Human BBB Models

Effects of Retinoic Acid (ATRA) in BBB Models

ATRA induces maturation in the stem cell-derived BBB model. ATRA has been reported to promote stem cell differentiation into mature cells through the regulation of multiple signaling pathways, including BMP9 and Wnt/β-catenin. In addition, retinoic acid can further promote cellular maturation by upregulating the expression of mature cell markers (e.g., ALB, CK18, and UGT1A) and downregulating the expression of immature markers (e.g., AFP and DLK). When retinoic acid was added to an in vitro BBB model, intercellular connectivity was enhanced, tight junction protein expression was promoted, and BBB integrity was improved.

These findings reveal a strong maturation-promoting effect of retinoic acid in the stem cell-derived BBB model.

Advantages of the Retinoic Acid-Enhanced Model

• Overcomes deficiencies in barrier tightness, transporter expression, and functional relevance.
• Enhanced barrier tightness closely mimics the low drug permeability of the in vivo BBB.
• Correct expression of transporters allows detailed study of drug transport mechanisms across the BBB.
• Provides high accuracy and reliability for drug permeability testing.
• Suitable for toxicity screening due to improved physiological relevance.

Applications of Co-Culture and Multicellular BBB Models in Drug Testing

At Creative Biolabs, we leverage advanced co-culture and multicellular BBB models to support comprehensive drug testing and screening, providing reliable and physiologically relevant data that accelerate your drug discovery projects.

As co-culture and multicellular BBB models continue to develop, we can expect to see them being used much more in drug testing. By being able to more accurately model the in vivo environment in vitro, complex models like these will be able to increase the efficiency and success of CNS drug development.

For your future CNS drug discovery projects, contact Creative Biolabs today and let us assist you in your drug discovery efforts with our advanced BBB models.

References

1. Park, Jun Sung, et al. "Establishing Co-Culture Blood–Brain Barrier Models for Different Neurodegeneration Conditions to Understand Its Effect on BBB Integrity." International Journal of Molecular Sciences, vol. 24, no. 6, Mar. 2023, p. 5283. DOI.org, https://doi.org/10.3390/ijms24065283.
2. Distributed under Open Access license CC BY 4.0, without modification.

Created July 2025

For Research Use Only. Not For Clinical Use.