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The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.
The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB's role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.
The BBB is a highly selective barrier that separates the CNS from the circulating blood. It protects critical brain functions from potentially disruptive substances, factors, and xenobiotics while also allowing the influx of nutrients and other metabolites required to maintain brain homeostasis1. The BBB is a multicellular NVU in which pericytes, astrocyte endfeet, and neuronal processes directly contact brain microvascular endothelial cells (BMECs). These interactions allow BMECs to form specialized barrier properties that are supported by tight and adherens junctions2,3. The formation of this barrier limits the paracellular passage of molecules, but it contains polarized transporters to actively transport molecules into the CNS or back into the blood1. Due to these unique barrier properties, the BBB constitutes a major obstacle to the delivery of biopharmaceuticals into the brain, and it is estimated that less than 5% of FDA-approved small molecules can reach the CNS4.
Animal models have been widely used to study BBB penetrance and the molecular mechanisms involved in BBB development5. While animal models faithfully represent the complex multicellular in vivo environment, differences in expression and activity of BBB transporters as well as substrate specificity across species often preclude accurate extrapolation of animal data to humans6. Thus, human-based models are critical to study the human BBB and for use in the development of drugs designed to target the CNS. This need becomes even more apparent with the increasing dominance of biological, human-specific drugs in the pharmaceutical development field. Accumulating evidence suggests that a compromised BBB is associated with a number of severe CNS disorders, including brain tumors and neurological diseases7,8,9. Human models faithfully reflecting these diseases have the potential to both 1) identify novel pathways that could be targeted for drug development and 2) predict CNS penetrance, thus reducing time and resources in preclinical studies and possibly decreasing failure rate in clinical trials.
In vitro models have been widely implemented to study interactions between BMECs and other cells of the NVU and conduct screens for prospective BBB-permeable drugs10. To recreate key aspects of the human BBB, in vitro models must display physiologically relevant properties (i.e., low paracellular permeability and physiologically relevant transendothelial electrical resistance [TEER] across the endothelial monolayer). In addition, the molecular profile of an in vitro system must include expression of representative functional transport systems. Typically, in vitro models are composed of endothelial cells that are co-cultured on a semipermeable membrane with combinations of other NVU cells to enhance BBB properties11. This approach allows simple and relatively rapid assessment of barrier functionality and molecule permeability. Such cell-based BBB models can be established with animal or human cell sources, including cells isolated from surgical excisions or immortalized BMEC lines.
Recently, protocols to differentiate human pluripotent cells into BMECs were introduced as an attractive source for in vitro human BBB models12,13. Induced pluripotent stem cell (iPSC)-derived BMECs (iBMECs) are highly scalable, demonstrate crucial morphological and functional characteristics of the human BBB, and carry the genetics of the patient. In culture, iBMECs form a monolayer that expresses tight junction markers and displays in vivo-like tight junction complexes. These cells also express BBB markers, including the BBB glucose transporter, glucose transporter 1 (GLUT1). Importantly, and unlike other alternative cell sources for human BMECs, iBMECs acquire barrier properties with values as high as those measured in vivo14, polarize along the basolateral axis, and express functional efflux pumps. Furthermore, the use of iPSCs from various subjects both 1) welcomes the opportunity to test aspects of the BBB in a personalized medicine manner and 2) provides a flexible source for generating additional cell types of the NVU. Generating these cells from an isogenic cell source to create personalized BBB chips would also aid in understanding inter individual differences in drug responses, which is a major cause for resistance or compromised response to treatment observed in clinical studies.
Use of iBMECs as monolayers in a dish or on a semi permeable transwell insert represents a powerful approach for BBB modeling. These systems tend to be robust, reproducible, and cost-effective. In addition, functional analyses such as TEER and permeability are relatively simple to perform. However, two-dimensional (2D) systems fail to recapitulate the 3D nature of in vivo tissue, and they lack the physiological shear stress forces provided by circulating blood and blood cells. This limits the ability of the vascular endothelium in these models to develop and maintain intrinsic BBB properties and functions.
Microengineered systems lined by living cells have been implemented to model various organ functionalities in a concept called organ-on-chips. By recreating in vivo-like multicellular architecture, tissue-tissue interfaces, physicochemical microenvironments, and vascular perfusion, these microengineered platforms generate levels of tissue and organ functionality not possible with conventional 2D culture systems. They also enable high resolution, real-time imaging, and analysis of biochemical, genetic, and metabolic profiles similar to living cells in the in vivo tissue and organ context. However, a particular challenge of the organ-on-chip is that the design, fabrication, and application of these microengineered chips requires specialized engineering expertise that is usually lacking in biologically oriented academic labs.
We have recently combined iPSC and organ-on-chip technologies to generate a personalized BBB chip model15,16. In order to overcome the technological challenges described, the commercially available Chip-S1 is used together with the culture module, an instrument designed to automate the maintenance of the chips in a simple and robust manner (Emulate Inc.). The BBB chip recreates interactions between neural and endothelial cells and achieves physiologically relevant TEER values, which is measured by custom made organ chips with integrated gold electrodes17. Additionally, the BBB chip displays low paracellular permeability, responds to inflammatory cues at the organ level, expresses active efflux pumps, and exhibits predictive transport of soluble biomarkers and biopharmaceuticals. Notably, BBB chips generated from several individuals captures the expected functional differences between healthy individuals and patients with neurological diseases15.
The protocol detailed below describes a reliable, efficient, and reproducible method for the generation of human iPSC-based BBB chips under dynamic flow conditions. Guidance is provided on the type of assays and endpoint analyses that can be performed directly in the BBB chip or from sampling effluent. Thus, the protocol demonstrates the spectrum of techniques that can be applied for evaluating biological and functional properties and responses in a human-relevant model.
A brief description of the iPSC-based BBB chip is provided here. Human iPSCs are initially differentiated and propagated in tissue culture flasks as free-floating aggregates of neural progenitors, termed EZ-spheres. The top channel of the Chip-S116,18,19 is seeded with dissociated EZ-spheres that form the "brain side" of the chip, as cells differentiate over 7 days into a mixed culture of neural progenitor cells (iNPCs), iAstrocytes, and iNeurons. Human iPSCs are also differentiated in tissue culture plates into iBMECs. The bottom channel of the chip is seeded with iBMECs to form the "blood side" as they develop to form an endothelial tube (Figure 1). The porous extracellular matrix (ECM)-coated membrane that separates the top and bottom channels 1) permits the formation of cell-to-cell interactions between channels and 2) allows the user to run permeability assays and image cells in either channel using a conventional light microscope.
1. Generation of iPSC-derived neural progenitor cells (iNPCs)
2. Differentiation of iPSCs into iBMECs
3. Microfabrication of the organ chip
4. Chip preparation
5. Surface activation and ECM coating
6. Seeding the "brain side" channel and differentiating EZ spheres into mixed neural cultures
7. Seeding iBMECs into the bottom channel to generate the "blood side"
8. Initiation of flow
9. Blood-to-brain paracellular permeability assessment
10. Immunocytochemistry
Figure 6A,B,C represents a BBB chip seeded with EZ-spheres on the "brain side" top channel and iBMECs on the "blood side'" bottom channel. iBMECs were seeded first and allowed to attach overnight, after which EZ-spheres were seeded. Chips were then cultured under static conditions with daily media replacement for seven days. The BBB chip was then fixed using 4% PFA at RT for 10 min and washed 3x with DPBS. Immunocytochemistry was performed on the BBB chip...
The combination of organ-on-chip technology and iPSC-derived cells in the NVU holds promise for accurate modeling of the human BBB. Here, we provide a detailed protocol for simple and robust application of the recently published iPSC-based BBB chip16. An overview and timing of the seeding paradigm is shown in Figure 3. To obtain and maintain barrier functions that are suitable for BBB modeling, generating a homogenous iBMEC monolayer and retaining its integrity are cr...
Cedars-Sinai owns a minority stock interest in Emulate, the company that produces the study's microfluidic Organ chips. An officer of Cedars-Sinai also serves on Emulate's Board of Directors. Emulate provided no financial support for this research. Cedars-Sinai and Emulate have patents filed related to this work.
We would like to thank Dr. Soshana Svendsen for critical editing. This work was supported by the Israel Science Foundation grant 1621/18, the Ministry of Science and Technology (MOST), Israel 3-15647, the California Institute for Regenerative Medicine grant ID DISC1-08800, the Sherman Family Foundation, NIH-NINDS grant 1UG3NS105703, and The ALS Association grant 18-SI-389. AH was funded by Wallenberg Foundation (grant number 2015.0178).
Name | Company | Catalog Number | Comments |
Accutase | EMD Millipore | SCR005 | Dissociation solution |
B27 | Gibco | 12587010 | |
Bfgf | Peprotech | 100-18B | |
Chip-S1 | Emulate Inc | Chip-S1 | Organ-Chip |
Collagen IV | Sigma | C5533 | |
DAPI | Invitrogen | D3571 | |
Dextran-FITC | Sigma | 46944 | |
DMEM: F12 | Thermo Fisher Scientific | 31330038 | |
Donkey serum | Sigma | D9663 | |
Emulate Reagent 1 (ER-1) | Emulate Inc | ER-1 | |
Emulate Reagent 2 (ER-2) | Emulate Inc | ER-2 | |
Fibronectin | Sigma | F1141 | |
Glial Fibrillary Acidic Protein (GFAP) | Dako | Z0334 | |
GLUT-1 | Invitrogen | MA5-11315 | |
Glutamax | Life Technologies | 35050038 | Glutamine supplement |
hBDNF | Peprotech | 450-02 | |
KOSR | Thermo Fisher Scientific | 10828028 | |
Laminin | Sigma | L2020 | |
Matrigel | Corning | 354234 | Basement membrane matrix |
mTeSR1 | StemCell Technologies, Inc. | 85851 | |
NEAA | Biological industries | 01-340-1B | |
Nestin | Millipore | MAB353 | |
NutriStem | Biological industries | 05-100-1A | Alternate media |
PECAM-1 | Thermo Fisher Scientific | 10333 | |
Platelet-poor plasma-derived bovine serum (PPP) | Biomedical Technologies | J64483AB | |
Retinoic acid (RA) | Sigma | R2625 | |
S100β | Abcam | ab6602 | |
Steriflip-GP Sterile Centrifuge Tube Top Filter Unit | Millipore | SCGP00525 | |
Triton X-100 | Sigma | X100 | |
ZO-1 Monoclonal Antibody | Invitrogen | 33-9100 | |
βIII-tubulin (Tuj1α) | Sigma | T8660 | |
β-mercaptoethanol | Life Technologies | 31350010 |
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