(d).

Differentiation methods
Unguided strategy	Generation of brain organoids with mixed cell lineages of forebrain, midbrain, hindbrain, and retina, enabling the organoids to grow with minimum external interference High variability and heterogeneity	[11, 31, 95]
Guided strategy	Directed differentiation to generate brain region-specific organoids, such as cerebral cortex, hippocampus, midbrain, and cerebellum	[10, 13, 14, 119, 135, 136]
Fused culture technologies for integration of different regions of the organoids	More closely resembling the complexity of the brain in identity, architecture, and interaction manners enhanced the formation of microcircuits with the local excitatory neurons	[123, 124, 135]
Long-term propagation, storage, and regrowth following the frozen and thaw cycles	CRISPR-Cas9-based knock-in of the mutant KRASG12D allele into human colon APC−/− organoids	[115, 137]
Application of 3D printing technology in	Enabled an engineered organ to maintain the spatial arrangement	[39, 134, 138, 139]
Organoids-on-a-chip based approach to	Could remove the dead cells via connecting with an external pumping	[140]
Generate the tube-shaped epithelial organoids	System, extending tissue lifespan and enabling the colonization of organoid tubes with microorganisms to model the host–microorganism interactions
Generation of microglia cell-containing microglia cerebral organoids	Microglia were naturally developed in cerebral organoids and displayed similar characteristic ramified morphology as in normal fetal brains.	[106, 141]
Generation of microglia-containing hCOs (mhCOs)	Microglia-containing hCOs (mhCOs) were generated via overexpression of the myeloid-specific transcription factor PU.1 in cortical organoids. The mhCOs have become an efficient tool for functional investigation of microglia in neurodevelopmental and neurodegenerative disorders, such as AD	[108]