Table 2.

Potential challenges that hinder the applications of organoid in regenerative medicine

Unresolved issues over organoids-based therapies	Challenges/concerns	Further measurements
PSC clonal variation	Huge variabilities were reported in different PSC-derived organoids due to the variations existed in the different PSC lines including epigenetic profiles, gene/marker expressions, as well as unpredictable long-term genomic destabilities	To generate more PSC lines from a diverse pool of genetic/epigenetic backgrounds, and conduct long-term in vitro and in vivo rigorous functional characterisation of PSC lines and PSC-derived organoids. Widely accepted international guideline and standardized (well-characterized) protocols are urgently required to circumvent this issue
Size and architectural differences	Organoids are usually much smaller in size comparing to in vivo organs, mainly due to the constraint of culture conditions and lack of vascularization	Further optimization into the culture conditions using novel culture technologies (e.g. spinning bioreactors and orbital shakers) that could improve oxygenation and increase nutrient availability to the organoid centre incorporation of vascularization strategies into organoids
Reproducibility	Batch-to-batch variations that may negatively affect treatment outcomes and raise safety concerns	To develop experimental model systems with high fidelity, robustness of cell fate specification, and precision of self-assembly into higher-order organoids
Scalability	Large-scale production of functional organoids is still unachievable in current laboratory settings	Utilization of GMP-qualified PSC lines and products, standardized and scalable protocols, as well as stepwise quality control measurement
Inadequate morphology/function	Lack of cell-type diversity and communication leads to inadequate functional development	More cells (vascular and non-vascular) and environmental cues (haemodynamic response, hypoxia, nutrients, PH, electrolyte, etc.) could be incorporated into the experimental model systems to produce functional and dynamic organoids
Lack of pre-clinical validation	Currently, organoid-based pre-clinical studies are largely conducted on small animals	Examine the in vivo long-term patency, compatibility, and applicability in large animals with human disease settings
Limited understanding of cell types	Cell types in organoid models were currently validated using staining for known marker genes, which need to be more extensively characterized for it to be determined if they are truly reflective of the rich cell diversity found in the complex human organs	New technologies such as single-cell RNA-sequencing should be used to better define the molecular taxonomy of cell types within organoids
Lack of stable organized elastin	A lack of stable organized elastin in the vascular organoids is likely to contribute to poor in vivo patency and/or vascular rupture	New approaches in stimulating elastogenesis in the vascular organoids should be developed/incorporated into the current protocols, such as promoting de novo generation of elastic fibres by SMCs, incorporating engineered synthetic elastin into organoids, and using de-cellularized elastin-containing ECM
Unknown metabolic and physiological processes in organoids	Current studies are mainly focused on gene/protein expression, and have limitations in capturing the defining features of cellular identity/properties within organoids	More effective identification and comparisons of cellular composition in organoids using proteomics, epigenetic profiling, metabolomics, and electrophysiological characterization of cell types in organoids
Lack of knowledge into the interactions of organoids with other organs and the environment	A clear drawback of current organoid systems is lacking of inter-organ communication. Its interaction with local and remote environment remains elusive	More efforts are urgently required to develop an ‘organoid-on-a-chip’ technology

GMP, good manufacturing practices; SMCs, smooth muscle cells.