A Two-Dimensional Organoid Culture on Invasin or Basement Membrane Extract to Study Host-Bacteria Interactions

So we used human intestinal organoids to study how bacteria infects the gut and how gut cells respond to infection. We cultured the organoids in a 3D polarized monolayer to give bacteria access to the apical side of the cells. To begin, place the required materials inside a clean and sterile tissue culture hood.

Prepare a five microgram per milliliter solution of invasin in sterile PBS. Add 100 microliters of this solution to the upper compartment of a three micrometer pore polyester cell culture insert placed in a 24-well plate. Ensure that the membrane is completely covered and incubate overnight at four degrees Celsius.

Alternatively, if using basement membrane extract for coating, prepare a 10%solution in ice cold DMEM. Add 100 microliters of the diluted solution to the upper compartment of the three micrometer pore cell culture insert. Ensure complete coverage of the membrane surface and incubate for one hour at room temperature.

Remove the human intestinal organoids derived from colorectal cancer surgical waste from the incubator three to five days after splitting. Remove the spent medium. After resuspending the organoids in one milliliter of ice cold DMEM, transfer the suspension to a 15 milliliter conical tube.

Centrifuge the tube for five minutes at 500 G at four degrees Celsius, then, remove the supernatant completely. Resuspend the pellet in one milliliter of recombinant trypsin substitute. Pipette up and down using a P1000 pipette to mechanically disrupt the organoids.

Incubate at 37 degrees Celsius for 15 to 20 minutes until single cells or small clusters of three to four cells are obtained. Then, add five milliliters of ice cold DMEM, centrifuge for five minutes at 500 G at four degrees Celsius, and remove the supernatant. Resuspend cells in expansion medium at 100 microliters per cell culture insert.

Count cells using a hemocytometer and adjust the concentration to approximately two times 10 to the power of six cells per milliliter. Next, remove the coating solution from the inserts. Add 100 microliters of cell suspension containing two times 10 to the power of five cells to the upper compartment, and then dispense 500 microliters of expansion medium to the lower compartment.

Culture inserts at 37 degrees Celsius with 5%carbon dioxide until confluent, then differentiate with appropriate media and maintain for five days with regular media changes. Measure transepithelial electrical resistance of the cells with suitable electrodes. To begin, inoculate fluorescent adherent invasive Escherichia coli in three milliliters of Luria-Bertani medium inside a hood, and incubate overnight at 37 degrees Celsius with shaking.

After incubation, centrifuge 500 microliters of culture for five minutes at 1500 G.Resuspend the pellet in one milliliter of advanced DMEM supplemented medium. Measure the optical density of the suspension at 600 nanometers and dilute the cells to approximately one times 10 to the power of eight colony forming units per milliliter in advanced DMEM supplemented medium. Next, remove media in both compartments of the transwell harboring differentiated organoid monolayers.

Add 500 microliters of fresh differentiation medium to the lower compartment, and then introduce 100 microliters of bacterial suspension to the upper compartment for the infected condition. Add 100 microliters of advanced DMEM supplemented medium to the upper compartment of the control insert for the non-infected condition. Incubate the inserts at 37 degrees Celsius with 5%carbon dioxide and calculate the colony forming units per insert after plating on LB agar 2.5 hours post-infection.

At five hours post-infection, remove media and fix the cells with 4%paraformaldehyde inside a fume hood. Incubate the cells for 30 minutes and wash them three times with PBS. Excise the membrane insert and place cells facing upward in a well plate.

Stain with fluorescent phalloidin for one to two hours and wash three times with PBS. After staining the nuclei with DAPI for 10 minutes, wash the sample three times with PBS, mount it on a glass slide with mounting medium, and apply a cover slip. Then, seal the cover slip with nail polish.

Finally, acquire and process confocal Z-stack images to visualize nuclei, actin, and bacteria, including cross-sectional views. Assess epithelial response by extracting RNA and performing RT-qPCR to quantify gene expression. After reaching confluency, human intestinal cells on the cell culture insert displayed a mosaic-like pattern without visible gaps in the monolayer.

Transepithelial electrical resistance values above 500 ohms square centimeters were observed, consistent with the formation of a tight epithelial monolayer. The quantity of adherent invasive Escherichia coli in the basolateral compartment increased over time after infection. Confocal imaging demonstrated a polarized monolayer, with nuclei localized basally and an apical enrichment of the actin network.

Additionally, the bacterial cells were visualized. High magnification imaging revealed intracellular adherent invasive Escherichia coli and extracellular bacteria attached to the apical membrane. Tumor necrosis factor alpha expression was significantly upregulated in response to adherent invasive Escherichia coli infection compared to non-infected control.

This protocol allows researchers to study interactions between bacteria and differentiated non-tumoral intestinal human cells. So the most important consideration is to make sure that the organoid cells can reach confluence before infecting them with bacteria. Then you also need to make sure that the bacteria can grow properly during the infection.

This protocol can be adapted to study infection in other organs with different bacterial species and interactions between bacteria and different cell types.