Stem cell-derived neural organoids as platforms to investigate glioblastoma invasion and migration: A systematic review

Table 3 Neural organoids differentiation protocols

Ref.	Protocol	NOs phases	Plate	Medium	Supplements	Factors	ECM used	Time of each phase
Van De Looverbosch et al[33]	N/A	2D hiPSCs neural induction	N/A	Neurobasal + DMEM/F12 + Glutamax	Glutamax, MEM-NEAA, sodium pyruvate, 2-ME, human insulin, N2s, B27s, PS	LDN-193189 and SB-431542	-	11 days
		2D NPCs maturation	N/A	Neurobasal + DMEM/F12 + Glutamax	Glutamax, MEM-NEAA, sodium pyruvate, 2-ME, human insulin, N2s, B27s, PS	-	-	19 days
		Organoid production	U-bottom 96-well1	-	-	-	-	7 days
Ferreira et al[34]	Unguided	EB formation	ULA U-bottom 96-well	mTeSR 1	Normocin	ROCKi	-	Not clear
		Neural induction	ULA U-bottom 96-well	DMEM/F12	Knockout serum replacement, MEM-NEAA, Glutamax, 2-ME	ROCKi and bFGF	-	Until EBs reached 400-600 μm
		Neural induction	ULA 24-well	DMEM/F12	N2s, MEM-NEAA, heparin	-	-	Until neuroepithelium cues appeared
		ECM embedding	ULA 6-well	Neurobasal + DMEM/F12	N2s, B27s without vitamin A, MEM-NEAA, Glutamax, 2-ME, human insulin, Normocin	-	Matrigel	4 days
		Neural differentiation/maturation	ULA 6-well on orbital shaker	Neurobasal + DMEM/F12	N2s, B27s with vitamin A, MEM-NEAA, Glutamax, 2-ME, human insulin, Normocin	-	-	Up to use in co-cultures (> 40 days)
Pedrosa et al[25]	Guided	EB formation/neural induction	ULA V-shaped 96-well	DMEM/F12	KnockOut serum, MEM-NEAA, Glutamax, 2-ME, PS	ROCKi, DM and, SB-431542	-	7 days
		Neural differentiation	96-well	Neurobasal-A	B27 supplement without vitamin A, Glutamax, and PS	bFGF and EGF	-	14 days
		Neural differentiation	ULA 6-well	Neurobasal-A	B27s without vitamin A, Glutamax, and PS	bFGF, EGF, BDNF and, NT-3	-	21 days
		Neural maturation	ULA 6-well	Neurobasal-A	B27s without vitamin A, Glutamax, and PS	-	-	Up to use in co-cultures (> 42 days)
Fedorova et al[35]	Unguided	EB formation	V-bottom 96-well2	mTeSR 1	-	ROCKi	-	Until EB were at least 400 μm in diameter
		Neural induction	ULA 24-well	DMEM/F12	N2s, Glutamax, MEM-NEAA and, heparin	-	-	6 days
		ECM embedding	6 cm dish	Neurobasal + DMEM/F12	Glutamax, MEM-NEAA, N2s, human insulin, 2-ME, B27s without vitamin A and, PS	-	Geltrex	4 days
		Neural differentiation/maturation	Spinning bioreactor	Neurobasal + DMEM/F12	Glutamax, MEM-NEAA, N2s, human insulin, 2-ME, B27s with vitamin A and, PS	-	-	Up to use in co-cultures
Bassot et al[36]	Guided	EB formation	Microwell plate	Serum-free medium	-	ROCKi	-	12-36 hours
		Neural induction	6-well on orbital shaker	Neurobasal + DMEM/F12 + Glutamax	B27s and MEM-NEAA	TGFβ/activin/Nodali and BMPi	-	4 days
		Neural differentiation	6-well on orbital shaker	Neurobasal + DMEM/F12 + Glutamax	-	EGF, bFGF, BMPi, GDNF, BDNF and iγ-secretase	-	17 days
		Neural maturation	PTFE membrane 6-well without agitation	Neurobasal + DMEM/F12 + Glutamax	-	GDNF, BDNF and iγ-secretase	-	Up to use in co-cultures
Goranci-Buzhala et al[37]	Guided	EB formation/neural induction	ULA 96-well	NIM	-	ROCKi		5 days
		ECM embedding	NR	Neurobasal + DMEM/F12	N2s, B27s without vitamin A, 2-ME, human insulin, L-glutamin and, MEM-NEAA	-	Matrigel	4 days
		Neural differentiation/maturation	Spinner flask	Neurobasal + DMEM/F12	N2s, B27s without vitamin A, 2-ME, human insulin, L-glutamin, MEM-NEAA	DM; SB-431542	-	Up to use in co-cultures (> 10 days)
Azzarelli et al[14]	Unguided	EB formation	ULA 96-well	EB-media (basal 1 plus supplement A)	-	ROCKi		5 days
		Neural induction	ULA 24-well	NIM (basal 1 plus supplement B)	-	-	-	2 days
		ECM embedding	6 cm dish	Expansion media (basal 2 plus supplement C and D)	-	-	Matrigel	3 days
		Neural differentiation/maturation	6 cm dishes on orbital shaker	Maturation medium (basal 2 plus supplement E)	Matrigel dissolved in the medium	-	-	Up to use in co-cultures (> 10 days)
Krieger et al[38]	Unguided	EB formation/neural induction	AggreWell	NIM	-	ROCKi	-	5 days
		ECM embedding	NR	Neurobasal + DMEM/F12 + Glutamax	1:1 mixture of (N2s, human insulin, L-glutamine, MEM-NEAA, 2-ME): B27s, L-glutamine and, PS	-	Matrigel	Not clear
		Neural differentiation/maturation	NR	Neurobasal + DMEM/F12 + Glutamax	1:1 mixture of (N2s, human insulin, L-glutamine, MEM-NEAA, 2-ME): B27s, L-glutamine and, PS	-	-	Up to use in co-cultures
Goranci-Buzhala et al[23]	Guided	EB formation/neural induction	ULA 96-well	NIM	-	ROCKi	-	5 days
		ECM embedding	NR	Neurobasal + DMEM/F12	N2s, B27s without vitamin A, 2-ME, human insulin, L-glutamin, MEM-NEAA	-	Matrigel	4 days
		Neural differentiation/maturation	Spinner flask	Neurobasal + DMEM/F12	N2s, B27s without vitamin A, 2-ME, human insulin, L-glutamin, MEM-NEAA	DM and SB-431542	-	Up to use in co-cultures (> 10 days)
Linkous et al[24]	Unguided	EB formation/neural induction	LA 96-well	NIM	-	ROCKi	-	6-7 days
		Rosettes formation	Matrigel-coated 6-well	NIM	-	-	-	11-14 days
		ECM embedding	NR	Neurobasal + DMEM/F12	N2s, B27s without vitamin A, 2-ME, human insulin, Glutamax, MEM-NEAA	-	Matrigel	4 days
		Neural differentiation/maturation	Spinning bioreactor or orbital shaker	Neurobasal + DMEM/F12	N2s, B27s with vitamin A, 2-ME, human insulin, Glutamax and, MEM-NEAA	-	-	Up to use in co-cultures (> 25 days)

¹Treated anti-adherence solution.

²Poly-hydroxyethyl methacryla-treated non-adherence.

NOs: Neural organoids; ECM: Extra cellular matrix; 2D: Two-dimensional; hiPSCs: Human induced pluripotent stem cells; N/A: Not applicable; DMEM: Dulbecco’s Modified Eagle Medium; MEM-NEAA: Minimum essential media non-essential amino acids; 2-ME: 2-mercaptoetanol; N2s: N2 supplement; B27s: B27 supplement; PS: Penicillin/streptomycin; NPC: Neural progenitor cells; EB: Embryoid body; ULA: Ultra-low attachment; NR: No reported; LA: Low attachment; ROCKi: ROCK inhibitor; bFGF: Basal fibroblast growth factor; DM: Dorsomorphin; EGF: Epidermal growth factor; BDNF: Brain-derived neurotrophic factor; NT-3: Neurotrophin-3; TGFβ: Transforming growth factor β; Nodali: Nodal inhibitor; BMPi: Bone morphogenetic proteins inhibitor; GNDF: Glial derived neurotrophic factor; NIM: Neural induction medium.