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

Summary

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.

Abstract

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.

Introduction

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 junctions^2,3. The formation....

Protocol

1. Generation of iPSC-derived neural progenitor cells (iNPCs)

1. Produce EZ-spheres from iPSC colonies as described below and as previously published^20,21,22.
   1. 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).
   2. Remove iPSC medium and replace with 2 mL of EZ-sphere medium [ESM; DMEM:F12 7:3 supplemented with 100 ng/mL basic fibroblast growth factor (bFGF), 100 ng/mL epidermal growth factor, 5 µg/mL heparin, and 2% B....

Results

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.......

Discussion

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.......

Materials

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