Prostate Organoid Assay: A Matrix Gel Ring-based Ex Vivo Culture Technique to Study the Differentiation Capacity of Prostate Epithelial Cells

Begin this procedure with preparation of mouse basal and luminal prostate epithelial cells as described in the text protocol. Wash the cell pellet in 500 microliters of mouse organoid media. Then, resuspend the pellet at a cell density of 1,000 cells per microliter. To prepare master mixes, mix epithelial cells suspended in mouse organoid media with matrix gel to generate a final mixture that contains 25% cells in media and 75% matrix gel. Depending on the downstream application, basal cells are typically plated at a concentration of 100 to 2,000 cells per 80 microliters, whereas luminal cells are plated at a concentration of 2,000 to 10,000 cells per 80 microliters.

For each cell mixture, add 80 microliters of the matrix gel per well of a 24-well plate. Pipet a droplet onto the lower half of the wall of the well while avoiding direct contact with the poly-HEMA coating. After adding the matrix gel, swirl the plate to allow the matrix gel cell mixture to form a ring around the rim of the well. Place the 24-well plate into a 37 degrees Celsius 5% CO2 incubator, right side up, for 10 minutes to allow the matrix gel to partially harden.

After incubating for 10 minutes, flip the 24-well plate upside down and incubate for an additional 50 minutes to allow the matrix gel to completely harden. Then, add 350 microliters of pre-warmed mouse organoid media, drop-wise, to the center of each well. After adding the media, return the 24-well plate to the incubator.

To replenish mouse organoid media, tilt the 24-well plate at a 45-degree angle and gently remove existing media from the center of each well using a P1000 pipette while avoiding the matrix gel ring. Add 350 microliters of pre-warmed mouse organoid media as before. It is recommended to add a larger volume of media to organoids cultured for longer than five days to prevent rapid depletion of key nutrients and growth factors.