. 2024 Apr 18;9(3):865–890. doi: 10.1002/epi4.12941

TABLE 3.

Comparison of general and epilepsy related parameters studied using in vitro patient derived cell culture models, including appropriate methodologies (non neural cell cultures e.g. fibroblasts/blood cells, ²⁹ ^, ³⁰ ^, ³¹ ^, ³² ^, ³³ ^, ³⁴ iPSCs derived 2D neural cell cultures, ³⁵ ^, ³⁶ ^, ³⁷ ^, ³⁸ ^, ³⁹ ^, ⁴⁰ ^, ⁴¹ ^, ⁴² ^, ⁴³ ^, ⁴⁴ ^, ⁴⁵ ^, ⁴⁶ ^, ⁴⁷ ^, ⁴⁸ ^, ⁴⁹ iPSCs derived 3D neural cell cultures, ³² ^, ⁵⁰ ^, ⁵¹ ^, ⁵² ^, ⁵³ ^, ⁵⁴ ^, ⁵⁵ ^, ⁵⁶ ^, ⁵⁷ ^, ⁵⁸ ^, ⁵⁹ ^, ⁶⁰ ^, ⁶¹ direct reprogrammed neural cell cultures, ⁴⁴ ^, ⁶² ^, ⁶³ ^, ⁶⁴ resected human brain tissue cultures). ²⁵ ^, ²⁶ ^, ⁶⁵ ^, ⁶⁶ ^, ⁶⁷ ^, ⁶⁸ ^, ⁶⁹ ^, ⁷⁰ ^, ⁷¹ ^, ⁷²

General characteristics	Epilepsy‐related characteristics	Non‐neural cell cultures (fibroblasts/blood cells)	IPSCs‐derived 2D neural cell cultures	IPSCs‐derived 3D neural cell cultures	Direct reprogrammed neural cell cultures	Resected human brain tissue cultures
Molecular and gene expression profiling		YES Refs. [29, 30, 31, 32, 34]	YES Refs. [36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 73]	YES Refs. [50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61]	YES Refs. [44, 62, 63, 64]	YES Refs. [26, 67, 68, 69, 70, 71]
Detection of gene variants	Known or novel gene variants in epilepsy‐related genes	+	+	+	+	+
Variations in gene expression	Underexpression or overexpression of epilepsy‐related genes; neuronal excitability gene expression patterns specific for epilepsy; presence of specific proteins and ion channels (expression and function of ion channels associated with hyperexcitability)	+	+	+	+	+
Changes in cell signaling	Abnormal calcium signaling; activation and inhibion of the mTOR pathway	+	+	+	+	+
Available techniques		Next‐generation sequencing, Sanger sequencing, proteomics profiling, immunoblotting, qPCR, chemiluminescence	Next‐generation sequecing, Sanger sequencing, immunocytochemistry, immunoblotting, fluorescence microscopy, flow cytometry, qPCR, proteomics and transcriptional profiling, RNA‐Seq, ATAC‐Seq, calcium imaging, Sanger sequencing	Next‐generation sequecing, Sanger sequencing, immunohistochemistry, immunoblotting, fluorescence microscopy flow cytometry, qPCR, proteomics profiling, LC–MS/MS, RNA‐Seq, RNA, and DNA‐FISH	Next‐generation sequencing, immunocytochemistry, immunoblotting, fluorescence microscopy, flow cytometry	Next‐generation sequencing, immunohistochemistry, confocal microscopy, droplet‐based digital PCR
Morphology, proliferation, and cell type compositon		YES/NO Ref. [33]	YES Refs. [35, 36, 37, 39, 40, 41, 42]	YES Refs. [32, 51, 53, 54, 57, 58, 59, 61]	YES Refs. [62, 63, 64]	YES Refs. [26, 67, 68, 69, 70, 72]
Cell morphology	Soma size (dysmorphic neurons); spine density and morphology; dendritic overgrowth; synaptic markers; axonal sprouting	−	+	+	+	+
Cell proliferation and survival	Cell proliferation, abnormal neuronal and glial proliferation	+	+	+	+	+
The ratio of excitatory to inhibitory neurons	Increased excitatory activity; decreased inhibitory activity; altered exicatory/inhibitory balance in specific brain regions	−	+	+	+	+
Synapse and network formation	Abnormal synapse formation; synaptic markers; formation of aberrant neuronal circuits	−	+	+	+	+
Available techniques		Luminiscence, fluorescence	IFM, confocal microscopy, quantification of the dendrite bundles, axon initial segment imaging, neurite outgrowth assay, cell death assay, FRET, cell proliferation assay	IFM, confocal microscopy, synaptic puncta quantification, cell proliferation assay, morphometric analysis, radial glia‐like cells and heterogeneity analysis	IFM, confocal microscopy, morphometric analysis (areas, perimeters, and neurites features), proliferation assay, electron microscopy, apoptosis analysis	Immunohistochemistry, confocal microscopy, electron microscopy
Migration and development		NO	YES Refs. [41, 47]	YES Refs. [32, 57, 58, 59, 60, 61)]	YES/NO	YES Refs. [67, 68, 69, 70, 72]
Neuronal migration and development	Abnormal neuronal organization and connectivity (focal cortical dysplasia, lissencephaly, and heterotopia)	−	+	+	+/− (limited information)	+
Structural abnormalities	Gyral and sulcus patterns	−	−	+	−	+
Available techniques		−	IFM, cell migration assay, confocal microscopy, immunohistochemistry	IFM, confocal microscopy, neuronal migration assays, immunohistochemistry, qPCR	−	Widefield microscopy, immunohistochemistry
Energy metabolism		YES Refs. [29, 30, 31, 32, 33, 34]	YES Ref. [48]	YES Ref. [32]	YES Refs. [62, 63]	YES Ref. [71]
Mitochondrial function assessment	ATP levels; mitochondrial membrane potential; mitochondrial morphology; respiratory chain activity; reactive oxygen species production; and markers of mitochondrial biogenesis	+	+	+	+	+
Metabolic profiling	Increased glucose uptake and glycolytic activity; dysregulation of the TCA cycle; alterations in metabolite levels; impaired OXPHOS; neurotransmitter synthesis; alterations in lipid metabolism	+	+	+	+	+
Biomarker expression	Protein levels of mitochondrial enzymes; oxidative stress markers; glucose transporters; hexokinase; beta‐hydroxybutyrate; acetoacetate; glutamate/glutamine ratio; GABAergic biomarkers; lipid peroxidation products; metabolism of fatty acids	+	+	+	+	+
Available techniques		Spectrophotometry, respirometry, scintillation method, TEM, luminescence, IFM, flow cytometry, immunoblotting, biochemical techniques, qPCR, LC–MS/MS	Ceramide synthase assays, lipidomics	Enzymatic activity, thermal stability, and kinetic characterization, biochemical measurement	Mitochondrial membrane potential, network, and morphology, immunocytochemistry, determination of ROS, extracellular flux analysis, mitophagy analysis, flow cytometry	Spectrophotometric analysis, respirometry, mitochondrial translation assay
Electrophysiological properties		YES/NO Ref. [34]	YES Refs. [36, 37, 39, 40, 42, 43, 44, 45]	YES Refs. [51, 52, 54, 55, 61]	YES Refs. [44, 63, 64]	YES Refs. [25, 26, 65, 66, 72]
Cellular excitability	Hyperexcitability; changes in action potential firing rates; responses to external stimuli	−	+	+	+	+
Synaptic activity	Synaptic currents; neurotransmiter release; aberant synaptic activity	−	+	+	+	+
Neuronal networks activity	Disrupted synchronization of neuronal networks; aberrant network bursting activity; oscillations	−	+	+	+	+
Spontaneous electrical activity	Spontaneous neuronal firing; abnormal electrical activity; epileptiform discharges or bursts	−	+	+	+	+
Seizure‐like events (spontaneous or induced)	Ictal‐like discharges; paroxysmal depolarization shifts; spike‐and‐wave discharges; postictal depression	−	+	+	+	+
Response to drugs	Response to antiepileptic drugs and compounds targeting epileptogenic pathways	+	+	+	+	+
Available techniques		Respirometry	MEA, microarray analysis, optophysiology, intra/extra‐cellular recordings, ion selective electrodes, patch‐clamp, single‐cell electrophysiology, ATAC seq	MEA, optophysiology, intra/extra‐cellular recordings, ion selective electrodes, local field potential, patch‐clamp, cell‐attached recordings	MEA, microarray analysis, optophysiology, intra/extra‐cellular recordings, ion selective electrodes, patch‐clamp, single‐cell electrophysiology, ATAC seq	MEA, optophysiology, intra/extra‐cellular recordings, ion selective electrodes, local field potential, patch‐clamp, cell‐attached recordings

Abbreviations: 2D, two dimensional; 3D, three dimensional; ATAC Seq, assay for transposase accessible chromatin with high throughput sequencing; DNA FISH, DNA fluorescence in situ hybridization; FRET, fluorescence resonance energy transfer; IFM, immunofluorescence microscopy; iPSCs, induced pluripotent stem cells; LC–MS/MS, liquid chromatography tandem mass spectrometry; MEA, multi electrode array; OXPHOS, oxidative phosphorylation; qPCR, quantitative polymerase chain reaction; RNA‐Seq, RNA sequencing; ROS, reactive oxygen species; TCA, tricarboxylic acid cycle; TEM, transmission electron microscopy.