Table 1.

Comparison of methods for introducing microorganisms into organoids

Method	Description	Advantages	Disadvantages
Microinjection61,67,85,216,217	Injecting microorganisms into the lumen of organoids using a microinjection device.	1. The method preserves the structural integrity of the organoids, and microorganisms only contact the apical side of organoids, providing a more realistic gastrointestinal simulation environment, especially for the anaerobic bacteria. 2. The entire process of microbe-organoid interactions can be observed, including initial interactions and early host responses. 3. Quantitative experiments can be performed by controlling the MOI. 4. This method has no special requirements for organoid culture conditions and can be applied for most 3D organoids.	1. This method requires a very specialized setup and is lacking of a standard procedure for different organoids. 2. The manual nature of the microinjection process makes it difficult to apply to high-throughput experiments, and the sequential injections resulted in asynchrony of experimental exposures. 3. The leakage of injected microorganisms toward the basolateral side can influence the readout, and the closed lumen may cause nutrient and oxygen consumption and metabolites accumulation. 4. Injections of small volumes of material are often imprecise, and differences in organoid size as well as luminal contents may cause uneven distribution of the injected material.
Organoid-derived fragment or epithelial monolayers220–224	Linearizing 3D Organoids into 2D Systems such as extracellular matrix-coated dish. The organoid-derived monolayer contains various epithelial cell lineages and enables the introduction of microorganisms via direct addition to the culture media.	1. The accessibility of the apical side of organoids is enhanced and the introduction of microorganisms can be achieved with an easily applicable setup. 2. This method can be applied to high-throughput experiments and can effectively reduce group differences caused by irrelevant variables in comparison or screening experiments. 3. Long-term co-culture with anaerobic bacteria can be achieved by incubating organoids in an aerobic environment while maintaining the apical chamber of a Transwell insert in an anaerobic environment. 4. Combined with the air-liquid interface or the microfabricated collagen scaffold array of crypt-like invaginations, this method can partially reconstruct epithelial-mesenchymal interactions and the crypt-like and villus-like structures.	1. The inoculation process caused mechanical damage to the organoids, and 2D organoids cannot reflect the structural features of lumen. 2. Certain bacterial media such as tryptone-yeast extract-glucose (TYG) and brain heart infusion may be toxic to the monolayers during introduction. 3. The success rate of establishing functional epithelial monolayers varies between different donors, which may limit its applicability. 4. Optimization measures such as providing continuous nutrient replenishment, creating an anaerobic chamber for obligate anaerobic bacteria, combining the air-liquid interface and collagen scaffold technology are costly and time-consuming.
3D organoids with growing reversed polarity84,218,219	Making the apical surface evert to face the media and introducing microorganisms by adding them directly to the culture medium.	1. This method simplifies the introduction of microorganisms while maintaining the 3D structure of organoids. 2. Without extracellular matrix affecting distribution, suspended apical-out organoids can be synchronously exposed to experimental agents and microorganisms. 3. Suspended organoid culture can be divided into multiple wells for different experimental conditions, which is more suitable for high-throughput experiments.	1. This method does not guarantee complete polarity reversion, so it is difficult to distinguish between the apical and basolateral interactions. 2. The mucus can be easily washed out, making it easier for foreign substances to enter the organoids. 3. Transferring apical-out organoids to new media is a time-consuming and iterative process. 4. Apical-out organoids exhibit slower proliferation and accelerated differentiation, suggesting that some of the pathways have been altered and may interfere with host-microbe interactions.
Microfluidic platforms225–229	Microfluidic platforms, including organ-on-a-chip, HuMiX and GuMI, are micro-engineered systems generated based on industrial computer microchips by microfabrication methods. Microfluidic platforms can provide organoids with precisely programmed biomimetic microenvironments and introduce microorganisms into organoids with ease and precision.	1. This method enables microbial diversity in organoids by tuning chemical gradients, oxygen gradients, dynamic mechanical stress and even incorporating multiple cell types and connecting multiple tissue platforms. 2. Simulation of key characteristics of the human gut and reconstruction of the mucus layer provides a better model for studying microbe-host interactions. 3. Standardized and automated organoid-on-a-chip enables high-throughput experiments. 4. Long-term coculture can be achieved through the continuous supply of nutrients and scavenging of metabolites via the microfluidic platform.	1. The complexity of organ structures and the heterogeneity of individuals determine that this method cannot fully simulate the real situation, so the applicability of this method needs to be discussed. 2. This method integrates programming, biochemistry, biomechanics, materials science and other disciplines and requires the cooperation of multiple teams and platforms, thus greatly increasing the experimental threshold and cost.