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

1. Plating Sorted Prostate Epithelial Cells into Primary Mouse Organoid Culture —  TIMING: 2-3 H (Excluding Poly-HEMA-coated Plate Preparation)

NOTE: Plates are coated with Poly-HEMA to prevent 2D colony formation on the surface of the well beneath the matrix gel. Prepare Poly-HEMA-coated plates 1 day prior to plating sorted basal or luminal prostate epithelial cells into mouse organoid culture. Thaw 1 mL aliquots of reduced growth factor matrix gel, hereafter referred to as matrix gel, on ice 2 h prior to step 1.1. Y-27632 (ROCK inhibitor) should be added to mouse organoid media immediately prior to step 1.1. Perform steps 1.1-1.8 on ice.

1. Pellet the cells in 5 mL round-bottom tubes by centrifugation at 800 x g for 5 min at 4 °C and aspirate the supernatant.
2. Wash the cell pellet in 500 µL of mouse organoid media (Table 1).
3. Pellet the cells by centrifugation at 800 x g for 5 min at 4 °C and aspirate the supernatant.
4. Resuspend in mouse organoid media at a cell density of 1,000 cells/µL.
5. To prepare master mixes, mix epithelial cells suspended in mouse organoid media with matrix gel to generate a final mixture that contains 25% cells/media and 75% matrix gel. Basal cells are typically plated at a concentration of 100-2,000 cells/80 µL, whereas luminal cells are typically plated at a concentration of 2,000-10,000 cells/80 µL. The density of cells plated varies depending upon the day of anticipated material collection and the desired downstream application.
   NOTE: Chill appropriately sized tube(s) for expected master mix volume 5 min prior to master mix preparation. To ensure the matrix gel does not harden while handling, it is critical to chill the pipette tip by pipetting the matrix gel 3-4 times prior to transferring it to a new tube.
6. Add 80 µL of the matrix gel/cell mixture per well of a 24-well plate. Pipetting a droplet onto the lower half of the wall of the well, while avoiding direct contact with the Poly-HEMA coating is recommended. 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.
7. Place the 24-well plate into a 37 °C 5% CO₂ incubator right side up for 10 min to allow the matrix gel to partially harden.
   NOTE: Begin warming mouse organoid media at 37 °C immediately after placing the 24-well plate in the incubator.
8. After incubating for 10 min, flip the 24-well plate upside-down and incubate for an additional 50 min to allow the matrix gel to completely harden.
9. Add 350 µL of pre-warmed mouse organoid media dropwise to the center of each well.
   NOTE: To maintain the integrity of the matrix gel, it is critical to avoid the matrix gel ring while adding media.
10. After adding the media, return the 24-well plate to the 37 °C 5% CO₂ incubator.

2. Replenishing Mouse Organoid Media — TIMING: 10-15 Min Per 24-well Plate

NOTE: Existing media should be replaced with fresh media every 48 h. Before each media change, pre-warm mouse organoid media. It is not necessary to add ROCK inhibitor to the media used for replenishing.

1. Tilt the 24-well plate at a 45° angle and gently remove existing media from the center of each well using a P1000 pipette while avoiding the matrix gel ring.
2. Add 350 µL of pre-warmed mouse organoid media as in step 1.9. It is recommended to add a larger volume of media (up to 1 mL) to organoids cultured for longer than 5 days to prevent the rapid depletion of key nutrients and growth factors.

Table 1: Instructions for the preparation of mouse organoid media