Generation of a Mouse Prostate Organoid-Based Model for Studying Host-Pathogen Interactions

This research focuses on understanding how bacteria pathogens infect the prostate epithelium, and which host pathogen interactions determine the infection outcome. Existing in vitro models poorly captured the prostatic epithelial diversity. This model provides a physiologically relevant system that reflects the difference cell types present in the tissue.

To begin, obtain three-dimensional murine prostate organoids cultured on drops of extracellular matrix in a 24 well plate. Aspirate the medium from the desired number of wells and add 500 microliters of fresh culture medium to each well. Using a P 1000 pipette mechanically disrupt the extracellular matrix drop in each well.

Collect this suspension in a 15-milliliter conical tube with no more than two wells collected per tube. Add fresh culture medium to bring the volume to 10 milliliters. Gently invert the tube three to four times to mix and centrifuge at 450 G for five minutes at four degrees Celsius.

Aspirate the supernatant. Re-suspend the pellet in two milliliters of enzyme solution, prewarm to 37 degrees Celsius and incubate the suspension in a water bath at 37 degrees Celsius for 15 minutes. Using a P 1000 pipette mix the cell suspension every five minutes during incubation.

After incubation, add fresh culture medium to reach a final volume of 10 milliliters. Gently invert the tube three to four times to mix. Centrifuge at 450 G for five minutes at four degrees Celsius.

Aspirate the supernatant without disturbing the pellet. Re-suspend the pellet in 300 microliters of culture medium supplemented with 10 micromolar rock inhibitor. Using a neubauer counting chamber count the cells in the suspension.

Dilute the suspension to a concentration of 62, 500 cells per milliliter in culture medium supplemented with 10 micromolar rock inhibitor. Then seed 200 microliters of the suspension per well for a 48 well plate and 300 microliters per well for chambered micro slides. Incubate the plates in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.

After one day, check if most single cells have attached to the two dimensional culture surface. Replace the culture medium every two to three days and continue culture until confluency is reached within seven days. For inducing differentiation to enrich luminal cells, add 10 nanomolar of five alpha dihydrotestosterone or DHT to the cell suspension during seeding.

Streak the uropathogenic Escherichia coli strain, or UPEC onto an LB agar plate supplemented with 100 micrograms per milliliter ampicillin. Incubate the plate at 37 degrees Celsius for 16 to 24 hours. Using a sterile loop inoculate Three milliliters of LB broth supplemented with 100 micrograms per milliliter ampicillin with a single bacterial colony.

Incubate the culture statically at 37 degrees Celsius for 12 to 16 hours. Vortex the overnight bacterial culture. Measure optical density of the culture at 600 nanometers.

Then, calculate the volume of culture needed to achieve a starting optical density of 0.05 in three milliliters of broth. Add the calculated volume of bacterial culture to fresh ampicillin-supplemented LB broth. Incubate statically at 37 degrees Celsius for 24 hours.

After incubation, vortex the bacterial culture. Measure the optical density at 600 nanometers. Calculate the culture volume needed to achieve an optical density of one.

Centrifuge the required volume at 12, 000 G for two minutes at room temperature. Aspirate and discard the supernatant. Re-suspend the bacterial pellet in one milliliter of primus and free organoid culture medium without DHT.

Then, dilute the suspension with mouse prostate organoid culture medium to achieve a multiplicity of infection of 100. Aspirate the culture medium from the prepared organoid-based model. Add 200 microliters of culture medium containing bacteria to each well of a 48-well plate.

Incubate for one hour in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide. Aspirate the bacteria containing medium from the wells and wash the wells three times with fresh culture medium to remove extracellular bacteria. Next, add culture medium containing 50 micrograms per milliliter gentamycin and incubate for 30 minutes.

Aspirate the medium. Add fresh culture medium, supplemented with 10 micrograms per milliliter gentamycin for the remaining infection time. Use a confocal microscope to image the infected cells after fluorescent staining and quantify infected cells using a flow cytometer.

In the control condition without dihydrotestosterone most cells were cytokeratin five positive basal cells with only a few showing low CD 24 8 luminal expression. Supplementing with one nanomolar DHT produced mixed basal luminal populations, though CD 24 A stayed low. Increasing DHT to 10 nanomolar promoted differentiation into luminal cells, while maintaining some basal cells after seven days of culture.

This correlated with the quantification studies showing a decrease in cytokeratin five positive basal cells and an increase in CD 24A positive luminal cells with increasing DHT concentration over time. At zero hours post-infection, UPEC adhered to epithelial cells in high numbers. After gentamycin treatment for one hour, the number of UPEC-positive cells dropped sharply.

At 24 hours, infected cell numbers were similar to those at one hour. Confocal imaging at 24 hours showed intracellular bacteria continued to replicate forming community-like clusters. This protocol allows researchers to study bacterial infection dynamics and those pathogen interactions in a model that preserves epithelial cellular diversity.

Future studies can use this model to investigate diverse prostate diseases, including infections, cancer biology and test potential therapeutic compounds.