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

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.