hiPSC-derived Blood Brain Barrier (BBB) Model Construction Service

Fig. 1 Physiological structure of the
BBB and pathological change.¹

The blood-brain barrier (BBB) is a selective permeability barrier between the central nervous system and the blood. It is comprised primarily of brain microvascular endothelial cells (BMECs) and a variety of supporting cells that comprise the neurovascular unit (NVU), including pericytes, astrocytes, and neurons. The cerebrovascular barrier plays an important role in maintaining central nervous system (CNS) homeostasis and is involved in several key processes, including nutrient transport, waste removal, and regulation of fluid composition in the brain. However, its selective permeability can also present challenges for therapeutic intervention. Many drugs and therapeutic agents have difficulty crossing the BBB, which may limit their effectiveness in treating neurological disorders.

As human induced pluripotent stem cell (hiPSC) technology has progressed, protocols have been established to derive brain microvascular endothelial cells (BMECs), pericytes, and neural cells (astrocytes, neurons, microglia, etc.) Creative Biolabs is proud to offer hiPSC-derived BBB models to study BBB permeability and to predict the pharmacological availability of drugs, such as monoclonal antibodies, adeno-associated virus vectors, and peptide-conjugated antisense oligonucleotides.

Strategies

	Transwell System The transwell is one of the most utilized in vitro BBB models, primarily due to its simplicity, user-friendliness, and straightforward protocols. In monoculture, BMECs are seeded alone in the porous membrane, allowing researchers to assess their barrier properties and permeability. In co-culture, BMECs are seeded on the top of the porous membrane while astrocytes or pericytes are seeded at the bottom of the well. Triple or quadruple cultures more closely mimic the in vivo environment, enabling the investigation of cellular interactions, signaling pathways, and the impact of various substances on BBB integrity and function.
	BBB-on-chip Model The BBB-on-chip model is a microfluidic system designed to mimic BBB for research and drug screening. The BBB chip features two separated microfluidic channel layers that integrate a 2D layer of BMECs with a 3D brain microenvironment composed of pericytes, astrocytes, and neurons, enabling highly sensitive quantification of molecular distributions independently within each spatial compartment.

hiPSC-derived BBB Model Offer Unique Advantages

• Human origin

Unlike traditional animal models, hiPSC-derived BBB models are generated from hiPSCs, providing a more accurate representation of human physiology and the physiological mechanisms of disease, overcoming pathological differences between human and animals.

• Cellular complexity

These models can incorporate various cell types found in the BBB, including BMECs, pericytes, and astrocytes. This cellular diversity more closely mimics the in vivo environment and helps in understanding cellular interactions and functions at the BBB.

• Disease modeling

hiPSC technology allows the generation of BBB models from patients with specific neurological disorders. This enables researchers to study disease mechanisms and drug responses in a controlled environment that reflects the genetic background of the individual.

Key Applications Include

• Drug Screening and Development
  
  • Screening compounds
    hiPSC-derived BBB model allows researchers to assess the permeability of drugs, small molecules, and cytokines across the BBB, helping identify candidates that can effectively reach CNS.
  • Toxicity testing
    Evaluating the toxicity of drug treatments at different doses and multiple time points in a validated, physiologically relevant BBB model.
• Disease modeling
  
  • Neurological disorders and neurodegenerative diseases (e.g. Alzheimer's disease - AD)
    hiPSC-derived BBB models from AD patients can mimic the AD pathological microenvironment. They are helpful for studying the clearance of beta-amyloid (Aβ) across the BBB and for exploring potential therapeutic strategies aimed at protecting or restoring BBB integrity in neurodegenerative diseases.
  • Ischemic stroke (IS)
    In IS, the BBB is compromised, leading to the disruption of tight junctions in the brain and ion transfer. Human iPSC-derived BBB models can be utilized to investigate the inflammatory responses and neurorepair efficacy that occur during stroke, providing insights into potential neuroprotective therapeutic strategies.
  • Infectious diseases
    hiPSC-derived BBB models are critical to understanding how infection affects BBB integrity, the mechanisms of pathogen entry into the brain, and the resulting immune response, thereby aiding the development of targeted therapies for CNS infections.
  • Brain cancers
    By adding brain tumor spheroids to mature BBB models, we can study tumor invasion mechanisms and the role of the BBB in cancer progression and metastasis. In addition, these models can be used to evaluate the efficacy of drugs and therapeutic agents targeting brain tumors, helping to optimize treatment strategies to enhance drug delivery and efficacy.
• Transport mechanism study
  hiPSC-derived BBB models allow scientists to understand drug delivery to the brain, identify potential barriers to therapy, and improve the design of treatments for neurological diseases.

Common Assays With hiPSC-derived BBB Model

• TEER measurement

Trans-endothelial electrical resistance (TEER) measurement assesses the integrity and barrier function of the endothelial cell layer by measuring the electrical resistance across the cell monolayer, with higher TEER values indicating a tighter barrier and better BBB function; this is often considered the primary indicator of a functional hiPSC-derived BBB model.

• Permeability measurement

Permeability measurements analyze the ability of substances to cross the BBB model. This assay typically uses fluorescent dyes (e.g., FITC-dextran) or radiolabeled compounds of different sizes to assess the movement of molecules across the BBB.

• Receptor-mediated transcytosis assay

This assay examines the mechanisms through which substances cross the BBB via receptor-mediated transcytosis. In this context, specific ligands or nanoparticles conjugated with targeting moieties are introduced to the hiPSC-derived BBB model. This allows researchers to quantify transport through the barrier by measuring the uptake and passage of these compounds. This assay provides insights into the potential of targeted drug delivery strategies to enhance therapeutic efficacy in the central nervous system.

• Gene and protein expression

Immunocytochemistry, qPCR, and Western blotting can be used to visualize and quantify the expression levels of junctional proteins (such as OCLN, ZO-1, VE-cad), and membrane transporters and receptors including GLUT1, CERP; ABCA1, and LRP1, assessing the functionality and integrity of the BBB under various conditions.

• Inflammation assays

The cultures can be exposed to pro-inflammatory cytokines TNFα, IL-1β, and their combination for 24 h to simulate inflammation. After 24 h, TEER measurements provide a method to evaluate barrier function.

• Cell migration

The hiPSC-derived BBB model can be used to evaluate the movement and penetration of immune cells across the BBB under different conditions.

Reference

1. Simöes Da Gama, Coraly, and Mélanie Morin-Brureau. "Study of BBB Dysregulation in Neuropathogenicity Using Integrative Human Model of Blood-Brain Barrier." Front Cell Neurosci. 2022;16:863836. Distributed under Open Access license CC BY 4.0. The original image was modified.