Name: generation of a human ipsc-based blood-brain barrier chip
Uploaded: 2020-03-02T14:30:00
Description: Watch this Scientific Journal Video about Generation of a Human iPSC-Based Blood-Brain Barrier Chip at JoVE.com

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).

1. Generation of iPSC-derived neural progenitor cells (iNPCs)
<ol>
	<li>Produce EZ-spheres from iPSC colonies as described below and as previously published20,21,22.
	<ol>
		<li>Culture iPSC colonies to confluency on basement membrane matrix-coated 6 well plates (0.5 mg/plate) in mTESR1 or other commercial media (see Table of Materials).</li>
		<li>Remove iPSC medium and replace with 2 mL of EZ-sphere medium [...

<ol>
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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...

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 &#34;brain side&#34; 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 &#34;blood side&#34; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>EMD Millipore</td>
			<td>SCR005</td>
			<td>Dissociation solution</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bfgf</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip-S1</td>
			<td>Emulate Inc</td>
			<td>Chip-S1</td>
			<td>Organ-Chip</td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-FITC</td>
			<td>Sigma</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM: F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>31330038</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Emulate Reagent 1 (ER-1)</td>
			<td>Emulate Inc</td>
			<td>ER-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Emulate Reagent 2 (ER-2)</td>
			<td>Emulate Inc</td>
			<td>ER-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial Fibrillary Acidic Protein (GFAP)</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td></td>
		</tr>
		<tr>
			<td>GLUT-1</td>
			<td>Invitrogen</td>
			<td>MA5-11315</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td>Glutamine supplement</td>
		</tr>
		<tr>
			<td>hBDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>KOSR</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>StemCell Technologies, Inc.</td>
			<td>85851</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Biological industries</td>
			<td>01-340-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td></td>
		</tr>
		<tr>
			<td>NutriStem</td>
			<td>Biological industries</td>
			<td>05-100-1A</td>
			<td>Alternate media</td>
		</tr>
		<tr>
			<td>PECAM-1</td>
			<td>Thermo Fisher Scientific</td>
			<td>10333</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet-poor plasma-derived bovine serum (PPP)</td>
			<td>Biomedical Technologies</td>
			<td>J64483AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid (RA)</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>S100&#946;</td>
			<td>Abcam</td>
			<td>ab6602</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>33-9100</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;III-tubulin (Tuj1&#945;)</td>
			<td>Sigma</td>
			<td>T8660</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>31350010</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a human ipsc-based blood-brain barrier 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&#39;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.

Figure 6A,B,C represents a BBB chip seeded with EZ-spheres on the &#34;brain side&#34; top channel and iBMECs on the &#34;blood side&#39;&#34; 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...

Watch this Scientific Journal Video about Generation of a Human iPSC-Based Blood-Brain Barrier Chip at JoVE.com

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

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 ...

This protocol will demonstrate how differentiated induced pluripotent stem cells can be seeded on an organ-on-chip to generate a fully human, personalized, microfluidic blood-brain barrier, which can be used to predict central nervous system drug permeability and to study neurological disorders. Using a commercially available organ-on-chip, we will demonstrate how any biologically oriented lab can use this technology to generate a personalized, microfluidic blood-brain barrier-on-chip. Accumulating evidences suggest that BBB plays a role in neurological disease by generating personalized BBB chips derived from iPSCs of individuals with a genetic neurological disease.It will be possible to study the role of the BBB in health and disease. This method can also be useful for pharma companies, which can use this human BBB platform to screen for the penetrability of candidate neurological drugs into the human brain. The application of organ-on-chip technologies usually requires specialized engineering skills.Using commercially available platforms enable the application of this technology in any biologically oriented lab. To begin, bring the dish containing the prepared chips to the biosafety cabinet. Using a P200 pipette, gently wash both channels by adding 200 microliters of neural differentiation medium into the inlet and pulling the liquid from the outlet.Avoiding contact with the ports, carefully aspirate excess media droplets from the surface of the chip. Gently agitate the cell suspension to ensure a homogeneous cell suspension. To seed the iPSC-derived neural progenitor cells into the top channel to generate the brain side, add a P200 tip containing 30 to 100 microliters of cell suspension at the concentration of one times 10 to the six cells per milliliter to the top channel inlet, and gently release the tip from the pipette.Take an empty P200 pipette, depress the plunger, insert into the top channel outlet, and carefully pull the single-cell suspension through the tip. Transfer the chip to the microscope to check the seeding density and homogenous distribution of cells within the top channel. After confirming the cell density at 20%coverage, immediately place the chips in the incubator for two hours at 37 degrees Celsius.After that, wash away the cells that do not attach by adding fresh neural differentiation medium into the inlet and pulling the liquid via pipette from the outlet. Keep the cells under static conditions at 37 degrees Celsius with a daily neural differentiation medium replacement. After iNPCs have attached or on a subsequent day following seeding, bring the dish containing the prepared chips to the biosafety cabinet.Gently wash the bottom channel with 200 microliters of endothelial cell medium. Avoiding contact with the ports, carefully aspirate droplets of excess endothelial cell medium from the surface of the chip, making sure to leave medium in both channels. Gently agitate the cell suspension to ensure a homogeneous cell suspension.Using a P200 pipette, draw up 30 to 100 microliters of the iPSC-derived brain microvascular endothelial cell suspension at the concentration of 14 to 20 times 10 to the six cells per milliliter, and place the tip into the bottom channel inlet. The most critical step to achieve functional barrier properties is the seeding of brain microvascular endothelial cells. It is crucial to seed cells at proper density to avoid bubble formation to verify homogeneous distribution within the organ chip.Depress the plunger on a P200 pipette with an empty tip, insert into the bottom channel outlet, and slowly release the pipette plunger to carefully pull the single-cell suspension through the bottom channel. Aspirate the excess cell suspension from the surface of the chip. Avoid direct contact with the inlet and outlet ports to ensure that no cell suspension is aspirated out of the channels.Transfer the chip to the microscope to check the seeding density. The bottom channel is filled with cells with no observable gaps in between. After confirming the correct cell density, seed the cells in the remaining chips.To attach the cells onto the porous membrane, which is located on top of the bottom channel, invert each chip, and let them rest in a chip cradle. Place a small reservoir containing sterile DPBS inside the 150-millimeter dish to provide humidity for the cells. Incubate the chips at 37 degrees Celsius for approximately three hours or until cells in the bottom channel have attached.Once the iPSC-derived brain microvascular endothelial cells have attached, flip the chips back to an upright position to allow cell attachment to the bottom portion of the bottom channel. 48 hours post-seeding of the iPSC-derived brain microvascular endothelial cells, equilibrate the temperature of the endothelial cell medium by warming in a 37-degree Celsius water bath for one hour. Then, degas the medium by incubation under a vacuum-driven filtration system for 15 minutes.Next, sanitize the exterior of the portable module packaging and trays with 70%ethanol, wipe, and transfer them to the biosafety cabinet. Open the package, and place the modules into the tray Orient them with the reservoirs toward the back of the tray. Pipette three milliliters of the pre-equilibrated, warm media to each inlet reservoir.Add endothelial cell culture medium to the inlet reservoir of the bottom channel and neural differentiation medium to the top channel inlet reservoir of the top channel. Then, pipette 300 microliters of the pre-equilibrated, warm media to each outlet reservoir, directly over each outlet port. Place up to six portable modules on each tray.Bring trays to the incubator, and slide completely into the culture module with the tray handle facing outward. Select and run the prime cycle on the culture module. Close the incubator door, and allow the culture module to prime the portable modules for about one minute.The priming cycle is completed when the status bar reads ready. Remove the tray from the culture module, and bring it to the biosafety cabinet. Inspect the underside of each portable module in the biosafety cabinet to verify that the portable modules were successfully primed.Look for the presence of small droplets at all four ports. If any portable module does not show droplets, rerun the prime cycle on those modules. If any media dripped onto the tray, which occurs more often by the outlet ports, clean the tray with 70%ethanol.Next, gently wash both channels of each chip with warm, equilibrated cell-specific culture medium to remove any possible bubbles in the channel, and place small droplets of media on the top of each inlet and outlet port. Insert the chips with carriers into the portable modules, and place up to six on each tray. Insert the trays into the culture module.Program the appropriate organ chip culture conditions, such as flow rate and stretch, on the culture module. As soon as the regulate cycle is complete, which takes approximately two hours, the programmed conditions start. After that, the culture module begins flow at the preset organ chip culture conditions.Immunocytochemistry on the iPSC-based blood-brain barrier chip seven days post-seeding shows the EZ spheres differentiated into a mixed neural cell population in the top brain side channel, including S100 beta-positive astrocytes, Nestin-positive neural progenitor cells, as well as GFAP-positive astrocytes, and beta III tubulin-positive neurons. IPSC-derived brain microvascular endothelial cells seeded in the bottom blood side channel expressed GLUT-1 and PECAM-1. Evaluation of the blood-brain barrier chip permeability demonstrates that the organ chip seeded with iPSC-derived brain microvascular endothelial cells and EZ spheres had a tight barrier compared to organ chips seeded with iPSC-derived brain microvascular endothelial cells alone.Organ chips seeded with EZ spheres alone did not display any barrier properties. From the initial surface treatment of the channel to the medium switching each day, avoid bubble formation in the channel. Throughout the step in the protocol in which surface activation reagent, extracellular matrix, or cell-containing medium needs to be inserted through the channels, it is extremely important to carefully verify that no bubble are found.The permeability assay can be performed to assess the human brain penetrability of candidate drugs. This approach can serve for testing potential therapeutics. The application of the iPSC-based BBB chip allows studying the role of the BBB in various neurological diseases.In the future, such platform may be useful for predictive, personalized medicine applications.

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...