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

The prostate epithelium primarily comprises basal epithelial and luminal epithelial cells. A monolayer of basal cells forms the outer lining of the gland and supports luminal cell survival. Conversely, the luminal cells line the lumen and secrete prostate-specific proteins.

To study prostatic epithelial differentiation ex vivo, begin with a suspension of basal prostate epithelial cells. Mix the suspension with a chilled basement membrane matrix in the desired ratio. Pipet this mixture next to the base of the inner wall of a cell-repellant culture plate. Swirl the plate for the mixture to form a ring along the circumference of the well.

Incubate the plate to allow the matrix to solidify. Supplement the culture with a preferred media. The growth factors in the media support the proliferation of basal epithelial cells. Concurrently, the cell-repellent nature of the culture dish prevents any cell attachment, encouraging the cells to form 3D spheroids within the matrix.

Over time, the spheroids develop lumens bordered with multi-layered epithelium. The lumen-lining basal epithelial cells eventually differentiate and form secretory luminal cells, giving rise to 3D glandular structures.

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.

• Prostate Epithelium - Consists of basal and luminal cells forming the prostate gland's structure.
• Basal Cells - Cells forming the outer lining of the prostate epithelium.
• Luminal Cells - Cells lining the lumen of the prostate gland and secreting prostate-specific proteins.
• 3D Spheroids - Cell structures formed in a cell-repellent environment.
• Organoid Assay - A method to study cell differentiation and formation of 3D glandular structures ex vivo.

• Introduction to Prostate Epithelium - It is formed of basal and luminal cells (e.g., prostate epithelial cells).
• Outline of Cellular Components - Basal cells support luminal cell survival; luminal cells line the lumen (e.g., organoid assay).
• Explanation of 3D Spheroids Formation - Grown within a matrix in a cell-repellent environment.
• Connection to Experiment - Formation of luminal cells from basal cells in 3D glandular structures.

• What are prostate epithelial cells and their role in the prostate epithelium?
• How does the organoid assay contribute to the study of prostatic epithelial differentiation?
• What is the process of 3D spheroids formation in a cell-repellent environment?

• Practical Applications - Used to study cell differentiation and formation of glandular structures ex vivo (e.g., organoid assay).
• Industry Impact - Crucial for biomedical research and development in urology (e.g., prostate health).
• Societal Importance - Contributes to better understanding and treatment of prostate diseases (e.g., prostate cancer).
• Scientific Advancements - Enabled the study of prostate cell proliferation and differentiation.