Blood Brain Barrier Permeability Assay2022-08-12T12:25:10-05:00
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  • The blood brain barrier (BBB) behaves as a highly selective semipermeable membrane separating circulating blood from the central nervous system while allowing the passage of glucose, water, and amino acids into the cerebrospinal fluid [1].
  • The BBB protects brain nervous tissue from the fluctuation of plasma composition, from pathogenic agents, and maintains homeostasis of the brain parenchyma by restricting non-specific flux of ions, peptides, proteins and cells into and out the brain.
  • The BBB is permeable to hydrophobic molecules and utilizes active transport to move ions, glucose, and other critical molecules across the membrane. When functioning properly, the blood brain barrier is a complex hurdle that must be overcome when delivering drugs to the brain.
  • Assessment of BBB permeability of drug compounds is critical for drugs targeting regions of the brain; many drugs developed to treat Central Nervous System (CNS) disorders are unable to reach the brain parenchyma in therapeutically relevant concentrations.
  • Poor penetration of the BBB is the cause for attrition for 95% of drugs developed for neurological disorders. As such, it is of great interest to explore potential molecules that can modulate BBB permeability [2].
  • Disruption of the BBB is observed in many neurological disorders, including multiple sclerosis, stroke, Alzheimer’s disease, epilepsy, and traumatic brain injury, and is frequently induced by neuroinflammation [1].
  • The BBB is a complex system, which is difficult to mimic in vitro, which typically had to be addressed with costly, low-throughput animal studies.
  • Utilizing a novel BBB in vitro model, we offer an in vitro assay capable of assessing the penetration kinetics of molecules passing across the BBB.
  • Additionally, we offer a complementary assay to assess modulation of BBB permeability due to drug treatment.
  • This assay service allows for both compound transport across the barrier to be studied as well as the effect of compounds on the structure and function of the BBB.

General Procedure

  1. 3D Blood Brain Barrier models are cultured over 4 days to establish a viable barrier as measured by transendothelial electrical resistance (> 150 Ω x cm2). On the fifth day, the models are dosed with test articles for six hours.
  2. If assessment of alterations to BBB permeability due to drug treatment is required, fluorescent dextran conjugate is added to wells.
  3. Media is sampled for quantitation of test articles by LCMS/MS, and for quantification of fluorescent probe by plate reader
  4. Apparent permeability is calculated

Figure 1. Brain Microvascular Endothelial Cells (Green) are cultured on the top of the transwell and Pericytes and Astrocytes are cultured (Orange and Blue) on the bottom of the transwell to form two distinct different cell layers.


Instrument(LC-MS/MS) system composed of an Agilent 1100 LC coupled with a SCIEX QTRAP 4000
Analysis MethodLCMS/MS
Markers70 kDa fluorescent dextran conjugate (for assessment of alterations to BBB permeability)
Cell modelHuman Blood Brain Barrier Model.

  • Brain Microvascular Endothelial Cells
  • Pericytes
  • Astrocytes
Time points6 hrs after dosing
(custom time points available)
Test Article ConcentrationSingle point assay (1 µM)
(custom concentrations available)
Number of Replicates3 replicates per time point
Quality Controls0.5% DMSO (vehicle control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryApparent Permeability of test compounds in either the apical or basal direction
Change in Apparent Permeability of BBB due to effect of drug compound (optional)

Validation Data

In Vitro vs In Vivo Outcomes

An in vitro model is only as useful as its ability to predict in vivo outcomes. With a handful of control compounds, we are able to demonstrate a positive relationship between the apparent permeability seen within in vivo systems to in vitro models.

Control Compounds

Within this model, we have demonstrated that caffeine (positive control) is able to pass through barrier whether or not the cells are present. A negative control is blocked from passing through barrier by the inclusion of the cells.

Positive Control

BBB Permeability

3.5 kDa fluorescently conjugated dextran is able to pass the barrier when cells are not present, however when cells are present dextran molecules are not able to pass through.

Reference Compounds

Reference compounds penetration across the BBB as a function  of time. 100 % indicates the compound has reached equilibrium across the membrane.


  1. Daneman, R. (2012). The blood–brain barrier in health and diseaseAnnals of neurology72(5), 648-672.
  2. Bourassa, P., Alata, W., Tremblay, C., Paris-Robidas, S., & Calon, F. (2019). Transferrin Receptor-Mediated Uptake at the Blood–Brain Barrier Is Not Impaired by Alzheimer’s Disease Neuropathology. Molecular pharmaceutics16(2), 583-594.

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