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

Table 1 Characteristics and methodology for the formation of glioblastoma tumor model (spheroids or cells)

Ref.	Tumor model (method)	Cell number	Cell type	Cell/stem cell	Fluorescent expressed	Culture medium	Supplements	Culture time (days)
Van De Looverbosch et al[33], 2025	Suspension cells	1000 and 2000	Patient-derived	GSCs (LBT037-EGFP)	EGFP	Neurocult medium	bFGF, EGF, heparin, and anti-anti	NR
Ferreira et al[34], 2024	Suspension cells	100000	Lineage	U343MG	GFP	DMEM	Anti-anti and FBS	NR
				LN18
Pedrosa et al[25], 2023	Spheres (grow up as tumor spheres in non-laminin coated plate)	NR	Patient-derived	Proneural (GIC7)	GFP	DMEM/F12	EGF, bFGF, glucose, N2, glutamine, BSA, and HEPES	NR
				Mesenchymal (PG88)
Fedorova et al[35], 2023	Spheres (poly-HEMA-treated ULA V-bottom 96-well plate + centrifuged (200 g/2 minute)	2000	Lineage	U87MG	GFP and tdTomato	DMEM/F12	Glutamax, NEAA, PS, and FBS	1
Bassot et al[36], 2023	Suspension cells	NR	Patient-derived	Classical GSC (Ge904)	Non-labeled	DMEM-high	Glucose, Glutamax, PS, and FBS	NR
Goranci-Buzhala et al[37], 2021	Suspension cells	1000	Patient-derived	GSCs (U3047MG (OPC-like)	mCherry	Neurocult NS-A	EGF, bFGF, N2, L-glutamine, B27 without vitamin A, heparin, and BSA	NR
				GSCs (U3024MG (MES-like)
Azzarelli et al[14], 2021	Suspension cells	10000 or 50000	Patient-derived	GSCs (GCGR-E27 and GCGR-E35)	H2B-GFP	D8437	EGF, bFGF, N2, Glutamax, B27, PS, and laminin	NR
Krieger et al[38], 2020	Suspension cells	1000	Patient-derived	4 patient-derived cell lines	GFP	Neurobasal medium	EGF, bFGF, L-glutamine, B27, and heparin	7-21
Goranci-Buzhala et al[23], 2020	Spheres (ULA U-bottom 96-well plate)	1000	Patient-derived	GSCs primary lines (U3047MG, U3024MG, 450)	mCherry	Neurocult NS-A	EGF, bFGF, N2, L-glutamin, B27 without vitamin A, heparin, and BSA	2
				GSCs recurrent lines (275-BIS)	GFP
Linkous et al[24], 2019	Suspension cells	10000	Patient-derived	GSCs	GFP	Neurobasal medium	bFGF, sodium, N2, B27, L-glutamine, and heparin	NR
					RFP-827

GSCs: Glioblastoma stem cells; EGFP: Enhanced green fluorescent protein; bFGF: Basal fibroblast growth fator; EGF: Epidermal growth factor; NR: Not reported; GFP: Green fluorescent protein; DMEM: Dulbecco’s Modified Eagle Medium; FBS: Fetal bovine serum; BSA: Bovine serum albumin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Poly-HEMA: Poly-hydroxyethyl methacryla; ULA: Ultra-low attachment; NEAA: Non-essential amino acids; PS: Penicillin/streptomycin; OPC-like: Oligodendrocyte-progenitor-like; MES-like: Mesenchymal-like; H2B-GFP: Histone-fused construct green fluorescent protein; RFP: Red fluorescent protein; Anti-Anti: Antibiotic/antimycotic.

Table 2 Characteristics of neural organoid models

Ref.	Cell/stem cell	iPSCs source	Cell number	NOs age (days)	Assessment size	Characterization1
Van De Looverbosch et al[33]	hiPSCs (iPSC0028)	Epithelium	10000	30	1077 ± 272 μm	NR
Ferreira et al[34]	hiPSCs (F9048)	Skin fibroblasts	10000	40	NR	SOX2, TUJ1, DCX
Pedrosa et al[25]	hiPSCs (BJiPSC-SV4F-9)	Skin fibroblasts	NR	41	NR	SOX2, TUJ1, GFAP, O4
Fedorova et al[35]	hiPSCs (MUNIi008-A)	Skin fibroblasts	2000-3000	55	NR	PAX6, TUJ1, DCX, MAP2, BRN2, SYN1
	hiPSCs (MUNIi009-A)
	hiPSCs (MUNIi010-A)
Bassot et al[36]	hiPSCs + hESCs (HS420)	Fetal skin fibroblasts + Blastocyst	1000	40	NR	TUJ1, MAP2, NeuN, GFAP, S100β, OLIG2
Goranci-Buzhala et al[37]	hiPSCs (IMR90)	Fetal lung fibroblast	35000	10	NR	NR
Azzarelli et al[14]	hiPSCs (IMR90)	Fetal lung fibroblast	9000	42	NR	SOX2, N-cadherin, PAX6, TUJ1, TBR1
Krieger et al[38]	hiPSCs (409b2)	Skin fibroblasts	1000	24	500-900 μm	PAX6, TUJ1
Goranci-Buzhala et al[23]	hiPSC (IMR90) + GSCs	Fetal lung fibroblast	35000 (hiPSC) + 1000 (GSC)	20, 40 and 60	500-700 μm	MAP2, SYN1
			35000 (hiPSC) + 5000 (GSC)
Linkous et al[24]	hESCs (WA01 and WA09)	Blastocyst	9000	NR	NR	Nestin, musashi-1, SOX2, PAX6 and TBR2
	hiPSCs (H6)	NR

¹Were considered only positive markers.

iPSCs: Induced pluripotent stem cells; NOs: Neural organoids; hiPSCs: Human induced pluripotent stem cells; NR: No reported; SOX2: Sex determining region box 2; TUJ1: Beta-tubulin III; DCX: Doublecortin; GFAP: Glial fibrillary acidic protein; PAX6: Paired box protein 6; BRN2: BEARSKIN2; SNY1: Synapsin1; hESCs: Human embryonic stem cells; MAP2: Microtubule-associated protein 2; NeuN: Neuron-specific nuclear; OLIG2: Oligodendrocyte transcription factor 2; S100β: Calcium-binding protein β; GSCs: Glioblastoma stem cells.

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.

Table 4 Analysis of the invasion of glioblastoma model into neural organoids

Ref.	Co-culture method	Co-culture time (days)	Groups evaluated	Invasion analysis method	Invasion analysis results	General purpose
Van De Looverbosch et al[33]	GSCs cells were plated onto the NOs	13	1000 GSCs	Fluorescence images and computing mapping (with clearing)	For both samples, most isolated GSCs were found dispersed between 25 and 200 μm from their surface	To demonstrate the value of computing mapping based on deep learning for counting cells in spheroids, identifying differentially labeled subpopulations in spheroids, and mapping the invasion of GBM cells into NOs
			2000 GSCs
Ferreira et al[34]	GBM cells were plated onto the NOs ULA 6-well plates with shaker (65 rpm)	14	Mock group (7 and 14 days)	Confocal fluorescence images and flow cytometry	Significant reduction in tumor cell proportion on ZIKV group and all CNS tumor cell lines reached the inner regions of the NOs after two weeks	To establish a co-culture model of human NOs with cancer cells from various CNS tumors and utilize these assemblies to investigate the oncolytic effects of ZIKV
			ZIKV infection group (20000 PFU) (7 and 14 days)
Pedrosa et al[25]	GBM cells or spheroids were plated onto the NOs	41	2000 GBM cells	Confocal fluorescence images and phase-contrast images	GFP-GICs started to integrate 24 hours after co-culture began. By 15 days, GFP-GIC7 and GFP-PG88 attached to and entered the Nos. By 41 days, both had successfully invaded and multiplied inside the NOs	To evaluate 5-ALA-mediated PDT for GBM treatment, aiming to eliminate infiltrating tumor cells while preserving normal tissue using GBM-initiating cells (GIC7 and PG88) in NOs
			2 tumor spheres
Fedorova et al[35]	GBM spheroids were placed on top of NOs (inclined at 45°)	30, 60, and 90	Inclined plane	Confocal fluorescence images (with clearing)	GBM migration into NOs increases over time. Migration distance and cell number are significantly higher at 60 and 90 days, with the highest number of migrating cells at 60 days. Matrigel and Geltrex increased GBM cell migration to NOs compared to the system without ECM. Matrigel showed a higher number of cells distant from the GBM/NOs border compared to Geltrex	To propose a GLICO model to study GBM growth and migration in NOs, highlighting the impact of ECM proteins
	GBM spheroids were placed of NOs embedded in a droplet of Matrigel or Geltrex in a ULA dishes with orbital shaker (0.035 g)		Matrigel
			Geltrex
Bassot et al[36]	GSCs cells were plated onto the NOs	5	Control (miR-ctrl)	Immunofluorescence and confocal imagens	Observed invasion of the GSCs into the NOs in the miR-Ctrl condition and a stronger signal for Ki-67 in the invasive single cells distant from their primary site, indicating that miRNAs can penetrate the NOs and affect GBM invasive capacity	To identify miR-17-3p, miR-222, and miR-340 as key regulators of GBM aggressiveness. Their combined modulation inhibits tumor growth, induces cell death, and shows therapeutic potential in GBM models
			Transfected (miR-17-3p, miR-222, miR-340)
Goranci-Buzhala et al[37]	GSCs cells were plated onto the NOs in a ULA-Lumox dish	20	Nek2-KD protein expressed with DOX	Immunofluorescence and confocal imagens (with clearing)	Nek2-KD-expressing GSCs failed to enter brain organoids. Naive cells diffused and recapitulated the known characteristics of invading GSCs, such as establishing protrusion-like processes in the form of microtubes. Nek2-KD-expressing U3047MG cells exhibited impaired invasion and failed to grow in NOs	To explore how GSCs suppress ciliogenesis to maintain self-renewal and tumor progression. Restoring cilia formation induces GSC differentiation, reducing tumor infiltration. The study suggests cilium induction as a potential therapy for GBM
			Naíve (control)
Azzarelli et al[14]	GSCs cells were plated in ULA 96-well plate onto NOs 6 cm dishes	7	10000 cells	Confocal fluorescence images	On low density, the GSCs group shows some individual cells dispersed sparsely across NOs, the invasion by interconnected streams was more prominent in high-density GSCs group. The behavior of the two cell lines was comparable (genetic profile, classified as RTKI and present PDGFRα amplification)	Followed the behavior of two different cell lines in the GOC system and found that GSCs with similar genetic alterations exhibit comparable behaviors upon organoid engraftment
			50000 cells
Krieger et al[38]	GBM cells were plated onto the NOs in a GravityTRAP-ULA + centrifuged (100 g per 3 minutes)	2	Patient F2	Immunohistochemistry and confocal images (with clearing)	To similar NOs size, the fraction of NOs volume taken up by tumor cells was similar across the 4 patient-derived cell lines, with similar sizes. Tumor cells spread widely in all cases. Invasion depths exceeded 100 μm in the majority, with some cells detected at 300 μm from the surface. Cells from patients F6 and F9 were less invasive than cells from F2 and F3	To investigate GBM invasion in a human-derived model using NOs as a scaffold. This model provides a clinically relevant platform for studying GBM
			Patient F3
			Patient F6
			Patient F9
Goranci-Buzhala et al[23]	GSCs spheres were plated onto the NOs	3-10	20 days-old organoids	Fluorescence (with clearing) and time-lapse images	GSCs integrated faster into older organoids, suggesting that mature NOs create a favorable environment for GSC growth, likely driven by neuronal activity and factors like NLGN3. Additionally, differences in invasive patterns were shown between primary and recurrent GSCs	To establish three different methods to assess GSC invasion in NOs and develop imaging techniques to validate organoids as reliable models. The assays reveal GSC affinities for organoids at different stages and capture key invasion aspects
			40 days-old organoids
			60 days-old organoids
Linkous et al[24]	GSCs cells were plated in 24-well plate onto NOs with shaker	14	Control group	Fluorescence (with clearing), luciferase activity and histopathological	GSC-827 exhibited infiltrative edges with tumor cells invading normal NOs, while GSC-923 showed a diffuse invasion pattern, resembling GBM patient samples. Both cell types (827 and 923) formed a network of tumor microtubes that facilitated infiltration into NOs	To train GLICO models for high throughput drug screening
			TMZ group
			BCNU group
			Ionizing radiation (0, 5 and 10 Gy)

GSCs: Glioblastoma stem cells; NOs: Neural organoids; GBM: Glioblastoma; ULA: Ultra-low attachment; ZIKV: Zika virus; PFU: Plaque-forming units; CNS: Central nervous system; GFP-GICs: Glioblastoma cell line with green fluorescent protein expression; 5-ALA: 5-aminolevulinic acid; PDT: Photodynamic therapy; GLICO: Glioma-cerebral organoid; ECM: Extracellular matrix; Ki67: Proliferation marker; Nek2-KD: Nek-knockdown; DOX: Doxycycline; RTKI: Receptor tyrosine kinase I; PDGFRα: Platelet-derived growth factor receptor alpha; GOC: Glioblastoma-organoid co-cultures; NLGN3: Neuroligin-3; TMZ: Temozolomide; BCNU: Carmustine.