Table 1.

Comparative analysis of experimental models for pathological oxidative stress in Central Nervous System tissues

	2D Cultures	Animal Models	Post-Mortem Human Tissue	Brain Organoids (3D Culture)
Advantages	Abundant culture methods and analytical techniques	Comparable size and anatomical structure	Accurate size and anatomical structure	Recapitulate 3D structural organization and diffusion of biological factors
	Can be generated from human iPSCs and ESCs	Can monitor the behavior of specific cortical cell types	Specific cortical cell types and precise developmental cues	Can be generated from human iPSCs and ESCs
	Widely used to study CNS disease progression	Widely available variants to study disease progression	Visible tissue degeneration in specific brain regions	Increasingly used to model human disease progression
	Can model near-physiological morphological changes and responses to oxidative stress	Can obtain useful measures of altered cognitive and behavioral states	Can obtain patient-specific measures of disease states	Capacity for self-directed organization and differentiation
	Highly scalable and high-throughput analysis of cell responses to biological factors	Can identify acute and chronic actions of reactive species during disease states	Can identify terminal pathological features of disease states across diverse human populations	Highly scalable and high-throughput analysis of cell responses to biological factors
	Can obtain functional cell- and tissue-specific information using simplified and low-cost methods	Can obtain functional whole-body 3D information i.e. systemic responses	Can identify some functional measures with a short “post-mortem interval”	Can obtain functional organ-specific 3D information i.e. electrophysiological network activity patterns
Limitations	Tissue composition and cell state change rapidly and demonstrate limited complexity	Significant metabolic, anatomical, and physiological differences to humans	Rapid biochemical changes during processing	Reliance on growth factors and differentiation protocols
	Poor representation of the in vivo physiological environment; limited cell-cell interaction	Lifespan of some species unaffected by high levels of oxidative stress; developmental differences	Loss of data on altered cell function and behavior due to tissue degeneration	Current limitations on functional and developmental neural cell maturation
	Lack of relevant data on cell-ECM or cell-scaffold interactions	Notable differences in brain mass, cellular organization, and regionalization	Decreasing donor/sample availability	Current methods are expensive, time-consuming, and characteristically provisional
	Automatically defined apical-basal polarization of cells	Results from these models often fail to translate to humans due to inter-species differences	Artifacts of neuronal death are rapidly introduced into dissected samples	Studies have reported stressful culture conditions and limited oxygen and nutrient diffusion
	Lack of 3D information; morphological constraints of 2D geometry	Greater neuronal density; lesser dendritic branching vs humans	Ethical and practical limitations of interrogating live/dead human brain tissue	Batch-batch or organoid-organoid variability in organization and “discrete” brain regions
	Risk of teratoma formation in stem-cell based therapy; limited differentiation capacity	Different patterns of age-related gene expression alterations	Poor study control to determine if observations/results are due to disease or caused by other agents	Lack of consensus for optimal culture conditions and methods to generate brain organoids