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 ...

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 Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

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 ...

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 ...