Co-culturing microorganisms on organoid-on-a-chip or tandem co-culture systems is a cutting-edge experimental technique that better simulates the microenvironment in vivo, allowing for the observation of complex interactions between microorganisms and host cells or other microorganisms. These culture systems are closer to physiological conditions and can provide dynamic fluid environments and microstructures that are difficult to achieve with traditional culture methods.

01

Materials and Equipment

1. Organoid-on-a-chip system

Microfluidic chip: A multi-chamber chip designed for co-culturing cells and microorganisms, equipped with fluid channels, embolism components, etc. (e.g., chips from companies like Future Mind, Bruina).

Pump system: Used to maintain fluid flow within the microfluidic chip, providing continuous nutrients and waste removal (e.g., peristaltic pumps or pressure-driven pump systems).

Organoids or cell lines: Organoids derived from patient or experimental models (e.g., intestinal, lung, etc.) or host cell lines (e.g., Caco-2 intestinal epithelial cells, A549 lung epithelial cells).

Microbial strains: Target microbial strains that need to be pre-cultured, such as gut symbionts (E. coli), pathogens (Salmonella), etc.

2. Media and Culture Media

Cell culture media: Media suitable for the growth of organoids or cells (e.g., DMEM, high-glucose media, etc.).

Microbial culture media: Media suitable for microbial growth (e.g., LB media, MRS media, etc.). Can be mixed or layered for different chambers.

Buffer solutions: Such as PBS, used for washing and removing unbound cells or microorganisms.

3. Experimental Equipment

Incubator: 37°C, 5% CO₂.

Microscope: For real-time observation of cells and microorganisms on the chip.

Fluid control system: Such as pressure pumps, flow rate regulators, etc.

Sterile workbench: For sterile operations.

02

Experimental Steps

1. Prepare the organoid-on-a-chip

Chip pre-treatment:

• Pre-treat the chip in a sterile environment. Depending on the chip design, it may be necessary to coat the surface with extracellular matrix (e.g., Matrigel, collagen, etc.) to assist organoid or cell adhesion and growth.

• Inject culture media into the chip and use a fluid pump or gravity flow system to ensure good circulation of the medium within the culture chambers.

Cell or organoid inoculation:

• Suspend organoids or cells in appropriate culture media, then inject them into the cell culture chamber of the chip using a sterile syringe or pipette.

• Allow to sit for several hours or overnight to enable cell adhesion or organoid stabilization within the chamber structure.

2. Microbial inoculation and co-culture

Microbial pre-culture:

• According to the experimental goals, first culture the target microorganisms in liquid media to reach the logarithmic growth phase (e.g., OD600 0.4-0.6) to ensure microbial viability.

• Select appropriate microbial culture media; some experiments may require separating the microorganisms from host cells or allowing contact in a flowing environment.

Microbial inoculation:

• Inject the microbial suspension into the microbial culture chamber of the chip. Depending on the design, microorganisms can directly contact the organoids or indirectly contact host cells through a semi-permeable membrane.

• If using a tandem culture system, first inject the microorganisms into a separate chamber, then connect that chamber to the organoid chamber via fluid flow to achieve dynamic co-culture.

Control fluid flow:

• Set flow rate: Adjust the fluid speed in the chip using the pump system, with a common flow rate range of 0.1-10 µL/min. The flow rate should be adjusted based on cell type and microbial growth characteristics to avoid washing away microorganisms or cells too quickly.

• Ensure that fluid interactions between the culture chambers can simulate physiological conditions, maintaining nutrient supply and waste removal.

3. Culture and Monitoring

Culturing conditions:

• Place the chip in a 37°C, 5% CO₂ incubator for continuous culture for several hours to days, depending on experimental needs.

• Regularly replace or supplement culture media to maintain good nutrient supply, especially during long-term cultures, ensuring no acidification or nutrient depletion occurs.

Real-time monitoring:

• Use a microscope (e.g., fluorescence microscope or confocal microscope) to monitor morphological changes of organoids or cells and the growth and infection status of microorganisms in real-time.

• Miniature sensors can be used to monitor changes in pH, oxygen concentration, and other parameters in the culture environment.

4. Data Collection and Analysis

1.Morphological observation:

Regularly photograph the morphology of organoids or cells using a microscope to observe the effects of microorganisms on host cells (e.g., cell apoptosis, barrier function changes, etc.).

2.Biomarker detection:

Samples can be taken (e.g., from the effluent in the chip) for further biological analysis:

qPCR or RT-PCR: Analyze gene expression changes in host cells or microorganisms.

ELISA: Detect inflammatory factors or metabolic products.

Flow cytometry: Analyze the survival rate or apoptosis of organoids or host cells.

3.Metabolite analysis:

Take effluent samples and analyze the concentration of microbial metabolites and their effects on host cells using techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS).

5. Termination of the experiment and chip cleaning

1.Terminate culture:

At the end of the experiment, stop the pump flow, remove residual culture media and microbial suspension from the chip, and fix cells and microorganisms for subsequent analysis (e.g., immunostaining or scanning electron microscopy observation).

2.Chip cleaning and reuse:

Most microfluidic chips are single-use, but if the chip supports multiple uses, it should be cleaned with appropriate cleaning solutions (e.g., enzymatic solutions, detergents) to remove residues and thoroughly disinfected.

03

Precautions

1. Sterile operations: Organoid-on-a-chip systems are very sensitive to contamination; all operations should be conducted in a strictly sterile environment.

2. Chip material selection: Choose chips made from different materials based on experimental needs; some chips may have adsorption or toxic effects on certain cells or microorganisms, and validation should be performed before experiments.

3. Fluid control: Flow rates that are too high or too low can affect the co-culture outcomes. It is recommended to conduct preliminary experiments to adjust the appropriate flow rate based on the characteristics of different cells and microorganisms.

4. Balance co-culture conditions: Host cells and microorganisms may have different requirements for culture conditions, especially regarding media selection and nutrient supply, which need to be designed reasonably.

⚠️Please note that the above steps need to be adjusted according to the specific conditions of the laboratory and the objectives of the experiment. In actual operations, multiple optimizations may be required to achieve the best experimental results, for learning reference only.