Table 2 Challenges related to the implementation of organoids in colorectal cancer research

Standardisation & Automation

- Clinical timeframe for PDOs generation variability.

- Lack of standardized and reproducible protocols for maintenance, culturing, cryopreservation.

- Maintaining viable co-culture conditions for various cell types, including the need for optimized culture media.

[19, 166,167,168,169]

Drug Screening

- Standardization of seeding density and organoid size for drug screening assays.

- Inter- and intra-experimental variability in high-throughput screening.

- No defined benchmark tests to assess drug efficacy/toxicity.

[19, 108, 168, 169]

Multi-Drug Combinations

- Drug-drug interactions and model variability.

- Difficult evaluation of drug activity and toxicity.

- Necessity to develop mathematical models to evaluate drug-drug interactions, dose selection and synergistic combinations.

[19, 176,177,178,179]

Complexity of Organoids

- Organoids lack the complexity of living organisms.

- Heterogeneity of tumors in phenotype, genotype, or cell composition complicates the use of combination therapies.

- Immunotherapies to be tested require more complex co-cultured model.

[108, 181, 183,184,185,186,187,188,189]

Mimicking Organ Architecture

- Mimicking organ architecture that conserves tissue specific characteristics.

- Lack of models that allow accurate in vivo situation like cell-to-cell interaction, migration.

[190, 191]