. 2023 Jun 26;15(6):632–653. doi: 10.4252/wjsc.v15.i6.632

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
Ref.	Cell origin	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 3-2: mTeSR1-cGMP	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
Wevers et al[20]	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)	iBMECs (10⁴) in NR medium	Astrocyte-neuron cells (1:4) (1.5 × 10⁴ cells/μL) in N2B27
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
				Days 3-1: mTeSR1-cGMP	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)
				Days 6-8: EC^+/+ (HESFM)	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)
		ACS-1024	LN 511-E8 or LN 411-E8 or collagen IV + fibronectin	HESFM	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
	Neural progenitors	EZ-Spheres	NA		B27 (2%) + vitamin A + N2 (1%) + hBDNF, (20 ng/mL)	iNPCs (10⁶ cells/mL) in NDM
	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
		CS03iCTR^mut^,²
		CS01iMCT8
		CS01iMCT8^cor^,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 4-3: mTeSR1-E8	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)

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

Modified 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.