Current overview of induced pluripotent stem cell-based blood-brain barrier-on-a-chip

Table 1 Microfluidic device design and fabrication

Ref.	Manufacturing					Characteristics of microdevices
	Fabrication	Main material of device	Technology used	Mold details	Membranes	Dimensions as width × height
Kurosawa et al[18], 2022	MIMETAS® (OrganoPlate® 3-lane plate)	Unspecified polymer	NR	NR	Membrane-free	Top and bottom channels: 320 μm × 220 μm. Gel channels: 360 μm × 220 μm. PhaseGuides®: 100 μm × 55 μm
Fengler et al[19], 2022	MIMETAS® (OrganoPlate® 3-lane plate)	Unspecified polymer	NR	NR	Membrane-free	Top and bottom channels: 320 μm × 220 μm. Gel channels: 360 μm × 220 μm. PhaseGuides®: 100 μm × 55 μm (width × height)
Wevers et al[20], 2021	MIMETAS® (OrganoPlate® 3-lane plate)	Unspecified polymer	NR	NR	Membrane-free	Top and bottom channels: 320 μm × 220 μm. Gel channels: 360 μm × 220 μm. PhaseGuides®: 100 μm × 55 μm
Noorani et al[21], 2021	Emulate® (brain on-a-chip)	PDMS	NR	NR	PDMS membrane	Brain channel: 1 mm × 1 mm. Blood channel: 1 mm × 0.2 mm
Middelkamp et al[22], 2021	In house	PDMS	Soft lithography	Material: PMMA. Fabrication: Micrommilling	PETE membrane (5 μm thick)	Straight bottom channel: 500 μm × 500 μm. Open-top compartment: 500 μm × 1500 μm
Choi et al[23], 2021	In house	PMMA	CNC	NA	PETE membrane	HUVEC microchannels: 800 μm × 200 μm. iBMECs microchannels: 800 μm × 500 μm
Motallebnejad et al[24], 2020	In house	NR	Soft lithography	NR	Membrane-free	800 μm × 100 μm
Lee et al[25], 2020	In house	PDMS	Soft lithography	Material: SU-8. Fabrication: Photolithography	Membrane-free	Fluidic channel: 1340 μm × 150 μm. Main channel: 2200 μm × 150 μm
Jagadeesan et al[26], 2020	In house	PDMS	Soft lithography	Material: SU-8. Fabrication: Photolithography	PDMS membrane (50 μm thick)	Top microchannel: 1 mm × 1 mm. Bottom microchannel: 1 mm × 0.2 mm
Vatine et al[27], 2019	In house	PDMS	Soft lithography	Material: SU-8. Fabrication: Photolithography	PDMS membrane (50 μm thick)	Top microchannel: 1 mm × 1 mm. Bottom microchannel: 1 mm × 0.2 mm
Park et al[28], 2019	In house	PDMS	Soft lithography	Material: Prototherm. Fabrication: 3D printed (Proto labs)	PE membrane (20 μm thick)	Hollow microchannels: 1 mm × 1 mm. Top channel: 1 mm × 1 mm. Bottom channel: 1 mm × 0.2 mm
Campisi et al[29], 2018	In house	PDMS	Soft lithography	Material: Silicon Wafer. Fabrication: NR	Membrane-free	Fluidic channel: 1000 μm × 150 μm. Main channel: 1300 μm × 150 μm. Distance between posts: 200 μm
Wang et al[30], 2017	In house	Objet VeroClear photopolymer	3D object printer (Objet 30Pro, Stratasys Ltd., Rehovot, Israel)	NA	PC membrane (0.4 μm pore size)	Main channel: 300 μm × 160 μm
DeStefano et al[31], 2017	In house	PDMS	Soft lithography	Material: Aluminum mold. Fabrication: NR	Membrane-free	390 μm, 450 μm, 550 μm, and 770 μm (different width)

3D: Three-dimensional; CNC: Computer numerical control; hBMECs: Human brain microvascular endothelial cells; HUVEC: Human umbilical vein endothelial cells; NA: Not applied; NR: Not reported; PDMS: Polydimethylsiloxane; PC: Porous polycarbonate; PE: Polyethylene terephthalate; PETE: Polyester track-etched; PMMA: Poly(methyl methacrylate); SU-8: Negative photoresist.

Table 2 Characteristics of the induced pluripotent stem cells used in the blood-brain barrier model, their cultivation and differentiation conditions

Ref.	Cell origin	Cell differentiation				BBB components model
		iPSCs line	Flask coating	Medium	Supplement	Differentiated cell/medium	Co-culture/medium
Kurosawa et al[18]	Human fetal lung fibroblast	IMR90-C4	Matrigel	Day 3-2: mTeSR1-cGMP	Day 3: With Y27632	iBMECs (107) in ESFM	NA
					Day 2: Without Y27632
				Day 0-5: UM	KOSR (20%) + glutamax (0.5%) + NEAA (1%) + β-mercaptoethanol (0.0007%)
				Day 6-8: EC+/+ (HESFM)	hPDS (1%) + RA (10 μM) + hFGF2 (20 ng/mL)
Fengler et al[19]	Human fetal lung fibroblast	IMR90-C4	Matrigel	Day 3-1: mTeSR1	Y27632 (10 μM)	iBMECs purified1 (106) in HBVP conditioned	NA
				Day 0-5: UM (DMEM/F12-HEPES)	Glutamax + KOSR + NEAA + β-Mercaptoethanol
				Day 6: EC+/+ (HESFM)	bPPP (1%) + RA (10 μM) + bFGF (20 ng/mL)
Wevers et al[20]	Human astrocytes	iPSCs	Geltrex	DMEM	FBS (10%) + N2 (1 ×) + P/S (1%)	iBMECs (104) in NR medium	Astrocyte-neuron cells (1:4) (1.5 × 104 cells/μL) in N2B27
	Human neural stem cells	Ax0018	Matrigel-GFR	Day 0-21: N2B27	BDNF (20 ng/mL) + GDNF (10 ng/mL) + AAc (100 μM) + db-cAMP1 (10 μM)
Noorani et al[21]	Human fetal lung fibroblast	IMR90-C4	NR	Day 1: E8	Y27632 (10 μm)	iBMECs (1.5 × 107 cells/mL) in NR medium	Primary ACs (106 cells/mL) and PCs (3.5 × 105 cells/mL) (3:1) in NR medium
				Day 0-6: UM	KOSR (20%) + NEAA (1%) + Glutamax (0.5%) + β-Mercaptoethanol (0.1 mM)
				Day 7-8: EC+/+ (HESFM)	bPPP (1%) + bFGF (20 ng/mL) + RA (10 μm)
				Day 9: EC-/- (HESFM)	Without bFGF and RA
Middelkamp et al[22]	Adult skin fibroblasts	GM25256	Laminin	Day 1: DMEM/F12	Primocin (0.1 mg/mL) + DX (4 μg/mL) + N2 (1 ×) + MEM-NEAA (1 ×) + NT3-RHP (10 ng/mL) + BDNF-RHP (10 ng/mL)	iNeurons (104) in E8	Rat ACs (1:1) and HUVECs (4 × 104) in ECGM-2
				Day 1 (after two hours): E8	RevitaCell (1 ×) + DX (4 μg/mL)
				Day 3-38: Neurobasal	Day 3: Primocin (0.1 mg/mL) + B-27 (1 ×) serum free + glutamax (1 ×) + DX (4 μg/mL) + NT-3 (10 ng/mL) + BDNF (10 ng/mL) + arabinoside hydrochloride (2 μm)
					Day 5: Refresh medium without arabinoside hydrochloride (2 μm)
					Day 9-38: Refresh medium with 2.5% fetal calf serum
Choi et al[23]	Human fetal lung fibroblast	IMR90-C4	Matrigel	Day 3: mTeSR1™-E8™	NR	iBMECs purified1 (1.2 × 107 cells/mL) in HESFM	AC (106 cells/mL) in EC-/-
				Day 0-5: UM (DMEM/F12)	KOSR (20%) + NEAA (100x) + glutamax (0.5%) + β-Mercaptoethanol (0.007%) (5% O2)
				Day 6-8: EC+/+ (HESFM)	Human serum (1%) + bFGF (20 ng/mL) + RA (10 μm) (5% O2)
				Day 9: EC-/- (HESFM)	Without bFGF and RA
Motallebnejad et al[24]	Human fetal lung fibroblast	IMR90-C4	Matrigel	Days 3-1: mTeSR1-cGMP	Day 3: Y27632 (10 μm)	iBMECs purified1 in NR medium	NA
					Day 2: Without Y27632
				Days 0-5: UM	NR
				Days 6-8: EC+/+ (HESFM)	Days 6-7: hPDS (1%) + RA (10 μM) + FGF2 (20 ng/mL)
					Day 8: bPPP (1%) or FBS (2%) + FGF (20 ng/mL) + RA (10 μM)
				Day 9: EC-/- (HESFM)	Without bFGF and RA
	Healthy human African American male from the bone marrow CD34+ cells	ACS-1024	LN 511-E8 or LN 411-E8 or collagen IV + fibronectin	HESFM	Day 8: bPPP (1%) or FBS (2%) + FGF (20 ng/mL) + RA (10 μM)
					Day 9: Without bFGF and RA
Lee et al[25]	Endothelial cells	hiPSC-ECs	Human fibronectin	VascuLife VEGF	iCell media supplement	hiPSC-ECs (6 × 106 cells/mL) in VascuLife VEGF with thrombin	PCs and ACs (106 cells/mL) in VascuLife VEGF with thrombin
Jagadeesan et al[26]	Neural progenitors	EZ-Spheres	NA	Day 1: EZ-sphere medium (DMEM/F12)	bFGF (100 ng/mL) + EGF (100 ng/mL) + heparin (5 μg/mL) + B27 (2%)	iNPCs (106 cells/mL) in NDM	ACs (9 × 105 cells/mL) and PCs (3 × 105 cells/mL) in in DMEM
					B27 (2%) + vitamin A + N2 (1%) + hBDNF, (20 ng/mL)
	NR	hiPSCs	Basement membrane matrix-coated	Day 1: mTeSR1	NR	iBMECs [(14-20) × 106 cells/mL] in EC-/- (HESFM)
				Day 2-8: UM (DMEM/F12)	KOSR (10%) + NEAA (1%) + glutamine (0.5%) + β-Mercaptoethanol (100 μm)
				Day 9-10: EC+/+ (HESFM)	bPPP (1%) + bFGF (20 ng/mL) + RA (10 μM)
				Day 11: EC-/- (HESFM)	Without bFGF and RA
Vatine et al[27]	Adult skin fibroblasts	CS03iCTR	Matrigel	Day 3: mTeSR1	NR	iNPCs (106 cells/mL) in NDM and iBMECs (1.4 × 104 cells/mL)	ACs (9 × 105 cells/mL) and PCs (3 × 105 cells/mL) in DMEM
		CS83iCTR
		CS03iCTRmut, 2
		CS01iMCT8
		CS01iMCT8cor, 2
	Peripheral blood	CS0172iCTR
		CS0188iCTR		Day 0: UM	Without bFGF
		CS0617iCTR
	Huntington’s disease	CS81iHD
Park et al[28]	Human fetal lung fibroblast	IMR90-C4	Matrigel	Day 3: mTeSR1	NR (5% O2)	iBMECs purified1 (2.3 × 107 cells/mL) in EC	ACs (7 × 105 cells/mL) and PCs (3 × 105 cells/mL) in ACM
				Day 0-6: UM (DMEM/F12)	KOSR (100 mL) + NEAA (5 mL) + glutamax (2.5 mL) + β-Mercaptoethanol (3.5 μL) (5% O2)
				Day 7-9: EC+/+	RA (5% O2)
Campisi et al[29]	Blood from 30-39-year-old healthy females	hiPSC-ECs	Human fibronectin	VascuLife VEGF	iCell media supplement + VEGF (50 ng/mL)	hiPSC-ECs (2 × 106 cells/mL) in EBM-2	Monoculture hiPSC-ECs (6 × 106 cells/mL) in EBM-2
							PCs (2 × 106 cells/mL) in EGM-2 MV
							PCs and ACs (2 × 106 cells/mL) in EGM-2 MV
Wang et al[30]	Human fetal lung fibroblast	IMR90-C4	Matrigel	Day 3: mTeSR1	NR	iBMECs in HESFM	Rat primary ACs in AGM
				Day 0-5: UM (DMEM/F12)	HEPES + KOSR (20%) + MEM-NEAA (1 ×) + L-glutamine (1 mM) + β-Mercaptoethanol (0.1 mM)
				Day 6-8: EC+/+ (HESFM)	hPDS (1%) + bFGF (20 ng/mL) + RA (10 μM)
DeStefano et al[31]	CD34 positive bone marrow	BC1-hiPSCs	Matrigel	Day 4-3: mTeSR1-E8	NR	iBMECs (105) in EC	NA
					Day 3: Y27632 (10 μm)
				Day 0-5: UM (DMEM/F12)	KOSR (20%) + NEAA (1%) + L-Glutamine (0.5%) + β-Mercaptoethanol (0.84 μm)
				Day 6-7: EC+/+ (HESFM)	hPDS (1%) + bFGF (20 ng/mL) + RA (10 μm)
				Day 8: EC (HESFM)	DB-cAMP (400 μm) or Y27632 (10 μm)

¹Induced pluripotent stem cell-derived brain microvascular endothelial cells were selectively expanded before to seed in the chip.

²Modified by clustered regularly interspaced short palindromic repeats. For human induced pluripotent stem cells-brain microvascular endothelial cells purification, day 8: ECM [1 μg/cm² LN 511-E8 or LN 411-E8 (iMatrix, iWAi), 1 μg/cm² full length laminin 511 (Biolamina), 100 μg/mL fibronectin (Millipore Sigma), or a mixture of 400 μg/mL collagen IV (Millipore Sigma) and 100 μg/mL of fibronectin] protein-coated ThinCert cell culture inserts (Greiner Bio-One), well plates or ibidi μ-slides. AT: Adipose tissue; BDNF-RHP: Brain-derived neurotrophic factor recombinant human protein; EGF: Epidermal growth factor; BM: Bone marrow; bPPP: Platelet-poor plasma derived bovine serum; CXCR4: C-X-C chemokine receptor type 4; db-cAMP1: 2’-O-Dibutyryladenosine-3’,5’-cyclic monophosphate; DMEM: Dulbecco’s modified eagle medium; E8 medium: Essential 8 medium; EGF: Epidermic growth factor; FDF2: Fibroblast growth factor 2; FL: Fetal lung; GFP: Green fluorescent protein; HESFM: Human endothelial serum free medium; hFGF2: Human fibroblast growth factor 2; hiPSC: Human induced pluripotent stem cell; HOXB4: Homeobox B4; hPDS: Human serum from platelet-poor human plasma; IB: Intrabone; IP: Intraperitoneal; IV: Intravenous; KOSR: Knockout serum replacement; Matrigel-GFR: Growth factor reduced matrigel; MEC: Brain microvascular endothelial cell; MEM: Minimum essential medium; NA: Not applied; NEAA: Non-essential amino acids; Nos2^-/^-: Deficient in type 2 nitric oxide; NR: Not reported; NT3-RHP: Neurotrophin-3 recombinant human protein; P/S: Penicillin/streptomycin; PDGFB: Platelet-derived growth factor subunit B; PPDS: Platelet poor derived serum; AAc: Ascorbic acid; SDF-1: Stromal cell-derived factor 1; solG-CSFR: Soluble granulocyte colony-stimulating factor decoy receptor; TPO: Thrombopoietin; UCB: Umbilical cord blood; MMP3: Matrix metallopeptidase 3.

Table 3 Applications of blood-brain barrier microfluidic three-dimensional models using induced pluripotent stem cells

Ref.	Application	Characterization	Evaluation technique	Outcomes
Kurosawa et al[18]	Build and evaluate a BBB 3D in vitro model	Capillary structure formation and tight junction proteins expression	Immunocytochemistry	Formation of the capillary structure, functional tight proteins; lower expression of ABC transporters than levels found in vivo, except for BCRP; expression of functional SLC transporters
		Transport proteins and receptors expression	Immunocytochemistry
			qPCR
		Tight junction functionality	Fluorescence (lucifer yellow and antipyrine)
			HPLC-MS/MS (test-drug transport)
		Transport proteins function	HPLC-MS/MS (test-drug transport)
Fengler et al[19]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	Capillary diameter CA. 40 times larger than in vivo brain vessels; physiologically relevant TEER values; physiologically similar localization of BCRP and GLUT-1 proteins. Promising BBB model for future drug screening tests
		Microvessel integrity	Fluorescence (DEX-A647 and sodium fluorescein)
		Microvessel permeability	Diazepam, Emricasan, Ac-YVAD-CMK, Z-DEVD-FMK, ZVAD (OH)-FMK, Staurosporine, and IL-1β
		Tight junction functionality	ELISA (Diazepam)
			TEER measurements
Wevers et al[20]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	Barrier functionality similar to that found in vivo; microfluidic model suitable for evaluating disruption of the BBB; successful ischemic stroke modeling. Potential use for modeling the BBB under sub-optimal conditions (disease) and for evaluating potential therapies
		Tight junction functionality	TEER measurements
		Microvessel permeability	Fluorescence (sodium fluorescein)
		Transport proteins expression	Fluorescence: P-gp inhibition
			qPCR
		Neuronal functionality	Calcium fluorescence imaging
	Ischemic stroke modeling	Microvessel permeability	Fluorescence (FITC-dextran)
		Mitochondrial membrane potential	Luminescence (CellTiter-GLO)
			ATP quantification
Noorani et al[21]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	BBB functionality remains intact for up to 7 d and is similar to that found in vivo; a more physiologically relevant BBB model; shear stress contributes positively to BBB tightness
		Microvessel permeability	UPLC-MS/MS: [13C12] sucrose and [13C6] mannitol
		Transport proteins expression	Immunocytochemistry
			Fluorescence: P-gp inhibition
Middelkamp et al[22]	Compare 2D cultures to microfluidic chip cultures	Neuronal differentiation and characterization of HUVECs	Immunocytochemistry	Culture in microfluidic chips promotes gene expression that more closely resembles that found in vivo
			RNA sequencing
			Transcriptomic analysis
Choi et al[23]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	cECMTE membrane with 10 m pores in microfluidic device were successful in mimicking the in vivo BBB, also allowing for cancer cell tissue migration. Promising BBB model for studying cancer metastasis, cell communication, and migration
			qPCR
		Tight junction functionality	Fluorescence (lucifer yellow)
			Transendothelial migration of cancer cells (CellMask)
			Immunocytochemistry
		Transport proteins expression	Immunocytochemistry
Motallebnejad et al[24]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	LM511-E8 ECM contributes to long-lasting endothelial cell and BBB function, in addition to promoting better shear stress responses. Authors recommend the use of LM511-E8 ECM for future studies involving BBB function
			Fluorescence (F-actin staining)
			qPCR
		Tight junction functionality	TEER measurements
			Fluorescence (rhodamine B-labeled neutral dextran)
Lee et al[25]	BBB permeability to polymer nanoparticles	Tight junction and transport proteins expression	qPCR	Fast analysis of polymer nanoparticles permeability; physiologically reliable BBB model
		Permeability to polymer nanoparticles	Fluorescence (polymer nanoparticles and FITC-dextran)
			3D fluorescence intensity maps
Jagadeesan et al[26]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	Successful fabrication of BBB model personalized for different human individuals; BBB models were able to mimic physiological differences between healthy and ill individuals
		Tight junction functionality and microvessel permeability	Fluorescence: FITC-dextran
		Transport proteins expression	Immunocytochemistry
		Neuronal differentiation
Vatine et al[27]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	Successful fabrication of BBB model personalized for different human individuals; BBB models were able to mimic physiological differences between healthy and ill individuals
			Transcriptional analysis
		Microvessel permeability and tight junction functionality	Fluorescence (FITC-dextran and 2NDBG)
			ELISA (human albumin, IgG and transferrin)
			LC-MS/MS (T3, colchicine, levetiracetam and retigabine)
			Transmission light microscopy
			TEER measurements
			Immunocytochemistry
		Transport proteins expression	Immunocytochemistry
			Transcriptional analysis
		Transport protein function	Fluorescence (rhodamine-123)
		Whole-blood neuronal toxicity	Colorimetric assay (quantification of lactic dehydrogenase)
		Neuronal functionality	Immunocytochemistry
			Calcium fluorescence imaging
Park et al[28]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	BBB functionality remains intact for up to 7 d. Promising BBB model for future drug and antibody transport studies
			Multiplex qPCR
			MS (proteomics)
		Tight junction functionality and microvessel permeability	Electron transmission microscopy
			TEER measurements
			Fluorescence (dextrans, cetuximab, angiopep-2, MEM75, 13E4)
			ELISA (dextrans, cetuximab)
		Transport proteins expression	Immunocytochemistry
			MS
		Transport proteins function	Fluorescence (rhodamine-123 and doxorubicin)
Campisi et al[29]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	Tri-culture of human iPSC-derived endothelial cells, astrocytes and pericytes spontaneously arranged into a BBB-like model. Promising BBB model for future preclinical experiments
			qPCR
		Tight junction functionality and microvessel permeability	Fluorescence (FITC-dextran)
		Characterization of astrocytes and pericytes	Immunocytochemistry
Wang et al[30]	Build and evaluate a BBB 3D in vitro model	Tight junction proteins expression	Immunocytochemistry	Pumpless media perfusion system that resembles the blood residence time within brain tissues; physiologically relevant TEER values maintained for up to 10 d. Promising BBB model for future drug permeability studies
		Tight junction functionality and microvessel permeability	TEER measurements
			Fluorescence: FITC-dextran and doxorubicin
			LC-MS/MS (caffeine and cimetidine)
DeStefano et al[31]	Evaluate BBB upon shear stress	Characterization of iPSC-derived endothelial cells morphology and function	Microscopy (time-lapse imaging analysis using ImageJ)	BBB endothelial cells display unique features that differ from endothelial cells from other tissues; shear stress plays a key role in BBB-like function in microfluidic models
		Tight junction proteins expression	Immunocytochemistry
			Western blot
			qPCR
		Transport proteins expression	qPCR

2NDBG: 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose; 3D: Three-dimensional; Ac-YVAD-CMK: Caspase-1 inhibitor; ATP: Adenosine triphosphate; UPLC-MS/MS: Ultra-performance liquid chromatography-mass spectrometry (tandem); BBB: Blood-brain barrier; BCRP: Breast cancer resistance protein; cECMTE: Condensed extracellular matrix track-etched; CellTiter-GLO: Luminescent cell viability assay; DEX-A647: Dextran conjugated to Alexa 647; ECM: Extracellular matrix; ELISA: Enzyme-linked immunoassay; FITC-dextran: Fluorescein isothiocyanate dextran; GLUT-1: Glucose transporter 1; hBMEC: Human brain microvascular endothelial cells; HPLC-MS/MS: High-performance liquid chromatography-mass spectrometry (tandem); HUVEC: Human umbilical vein endothelial cell; iBMEC: Induced pluripotent stem cell-derived brain microvascular endothelial-like cell; IgG: Immunoglobulin G; IL-1β: Interleukin 1 beta; iPSC: Induced pluripotent stem cell; LC-MS/MS: Liquid chromatography-mass spectrometry (tandem); LM511-E8: Fragment E8 of laminin 511; MS: Mass spectrometry; P-gp: Permeability glycoprotein; qPCR: Quantitative polymerase chain reaction; RNA: Ribonucleic acid; SLC: Solute carrier protein; TEER: Trans-epithelial electrical resistance; Z-DEVD-FMK: Caspase-3 inhibitor; ZVAD (OH)-FMK: Pan-caspase inhibitor.