Table 1 Current limitations of human organoids and suggested solutions to address drawbacks

Lack of microenvironment cells such as immune cells and stromal cells

Establishing co-culture of the organoids with microenvironment cells [6, 26]

Often limited in size and developmental state, analogous to embryonic/ foetal organs rather than adult organs

Integration of other organoids with the VOs or ECs, to enhance size, complexity, oxygen/nutrient distribution, and adult-like paracrine signalling [4, 6, 45]

Applying bioengineering methods and design principles to follow native organ multiscale structural orders and architecture [10, 16]

Often heterogeneous and irreproducible structures at macroscale (i.e. 100’s of microns to millimetres) [29]

Implementing techniques to control spatial and temporal organoid formation, making it more deterministic [31]

Developing differentiation protocols for region-specific organoids using microfluidics, matrix, or bioreactor approaches [30, 34]

Spontaneous morphogenesis processes within cell aggregates with minimal to no exogenous control [29]

Making it more automated and continual in situ monitoring of organoid behaviours [32] and guiding pattern by environmental cue [31]

Multisensory-integrated systems for automated and continual in situ monitoring of organoid behaviours [32]

Lack of inter-organ communication, fundamentally mimicking a part of the human body, not the entire body

Multiple organoids connection by a chamber device, ‘organoid-on-a-chip’ technology [10, 16, 35]. Compartmentalization of distinct organoid types enables fine-tuned organoid–organoid communication, while preventing their uncontrolled fusion [32, 33]

Variable cellular subpopulations, with unknown number and steps of intermediate cells particularly from hPSCs differentiation [37]

Developing a standardized differentiation protocol

QC for presence of unwanted cell types by single-cell RNA-sequencing and other analyses

Precisely controlling differentiation to desired cell types by blocking the formation of unwanted cell types or by overexpressing lineage-specifying transcription factors [37]

Lack of a standardized protocol or guidelines to ensure quality and reproducibility

Establishing standardized protocol with collective efforts by multi-Centre scientific consortium

Culturing under the well-defined culture media and ECM

Uncertainty in the composition of matrix used for 3D organoids generation

Developing mechanically and chemically defined synthetic extracellular matrices for organoids culture [41,42,43]

Low vascularization and maturity decrease organoid lifespan and increase necrotic core (nc) formation [4, 25]

Incorporating vascular organoids into other non-vascular organoids

Long-term culture, vascularization with microfluidics, and animal transplantation [34]

Relatively costly compared to 2d cultures, and fly, yeast, or worm models [8, 40]

Establishing widely accepted and used protocols for each type of organoid will decrease the cost progressively

Difficulty in cryopreservation and recovery

Developing new cryopreservation protocols and devices adapted for multicellular tissue banking and long-term storage