TABLE 3.

Limitations of current organoid systems and potential solutions for virus research (Shanks et al., 2009; Lehmann et al., 2019; Kim et al., 2020; Hofer and Lutolf, 2021).

Limitations	Organoid type	Points for prospective solutions
Limited week-scale lifespan	All, esp., ASC-derived	•Cryopreservation •Virus adaption •Organoid engineering
Maturation	All, esp., PSC-derived	•Maturation handling •Virus adaption •ASC-derived usage
Lack cell/system complexity	ASC-derived epithelial types	•Usage of PSC-derived to differentiate mesenchymal layer •Co-culture of cells/other organoids •Introduction of microbiota niche
Inaccessibility	Encapsulated organoids	•Increasing nutrient circulation through methods such as shaking and pumping •Cryopreserving at thriving status •Introduction of virus via injections or other physical poration •Develop apical-out epithelial organoids •Adopt 2D Transwell culture for simple cell layer
Limited reproducibility	Esp., ASC-derived	•Standardize resources for scaffolds and medium supplies •Standardize and simplify protocol •Use well-defined start materials for ASC-derived, and colonized PSC stocks •Subculture organoids from a cryopreserved organoid stocks
Readout limitations	Aggregated organoid capsules	•Imaging: (1) Use organoid microscopy or imager; (2) Adopt 2D Transwell culture; (3) Make organoids into continual sections •Molecular monitoring: implement biosensors •Computational modeling simulation •Omics profiling: Connect to various OMICS platforms, especially updates based on single cell throughput