The 3D organoid system is considered more physiologically relevant than 2D assays, and it can provide valuable information about the biology that would otherwise be challenging to acquire. This is an adaptable system where we can test pharmacological and genetic manipulations with less time and a significantly lower cost than using an in vivo approach. Matrix trial is very tricky to handle, as it solidifies when it reaches room temperature.
It can only be drawn through a pipette when it is liquid and must be maintained on ice. The integrity of the ring is important to maintain. If the structure is disrupted, that can negatively affect organoid growth, and you can easily lose material when changing media.
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 re-suspend 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. Pipette 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-degree Celsius, 5%C02 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. Remove the media from each well as before.
To collect organoids, repeatedly blast the matrix gel by pipetting one milliliter of pre-warmed dispase-containing media directly onto the matrix gel ring until the entire ring is dislodged. Transfer the dislodged matrix gel organoid mixture to a 1.5-milliliter micro-centrifuge tube. Following complete digestion as described in the text protocol, add phosphate-buffered saline to the organoid pellet, and re-suspend by gently flicking.
Pellet the organoids by centrifugation at 800 times G for five minutes at room temperature, and remove the supernatant using a micro-pipette. Re-suspend the organoid pellets in 100 microliters of protein lysis buffer per 10 microliters of packed cell volume. Flick to re-suspend.
Now, sonicate the organoids by submerging tubes in wet ice and gently applying the tip of the sonic dismembrator to the outside of the micro-centrifuge tube. Sonicate for 40 seconds at 20 kilohertz before proceeding to Western blotting with established protocols. To collect prostate organoids from the 24-well plates, remove the media from each well as before.
Digest the matrix gel by incubating with 500 microliters of dispase-containing media for 30 minutes in a 37-degree Celsius, 5%CO2 incubator. Collect digested organoid suspension in a micro-centrifuge tube. Then, pellet the organoids by centrifugation at 800 times G for three minutes at room temperature, and remove the supernatant.
To perform whole-mount immunofluorescent staining of prostate organoids, first add 500 microliters of 4%paraformaldehyde in PBS. Incubate the organoids for two hours at room temperature with gentle shaking. After washing the pellet as described in the text protocol, add one microgram per milliliter of DAPI stain in blocking solution, and incubate for two hours at room temperature.
Protect the sample from light during incubation from this step forward. Following centrifugation of the organoids as before, add primary antibody in blocking solution and incubate overnight at four degrees Celsius with gentle shaking. Pellet the organoids again, and wash the pellet with one milliter of PBS for 15 minutes with gentle shaking.
Repeat this washing procedure two times. Then add secondary antibody in blocking solution, and incubate overnight at four degrees Celsius with gentle shaking. Following incubation, pellet the organoids, and wash the pellet for an additional two times.
Add one milliliter of 30%sucrose in PBS with 1%Triton X-100 to the pelleted organoids. Then incubate for two hours at room temperature with gentle shaking. After pelleting the organoids again, add one millimeter of 45%sucrose in PBS with 1%Triton X-100, and gently shake for two hours at room temperature.
Then, repeat the procedure, except add one milliliter of 60%sucrose in PBS with 1%Triton X-100. Pellet the organoids by centrifugation at 800 times G for three minutes at room temperature, and remove 95%of the supernatant. Observe the pellet under UV light to confirm that it was not lost during removal of the supernatant.
The pellet becomes looser as the concentration of sucrose becomes higher. Transfer 10 to 20 microliters of the remaining suspension to a chambered coverslip, and proceed to confocal microscopy. Basal and luminal cells form organoids with distinct morphologies.
While most basal-derived organoids are similar in size after seven days in culture, luminal-derived organoids exhibit significant heterogeneity. Furthermore, most basal-derived organoids contain lumens surrounded by multilayered epithelium, whereas luminal-derived organoids range in morphology from hollow with single-layered epithelium to solid with multi-layered cords of cells that do not canalize. Western blot analysis revealed that basal and luminal-derived organoids retain features associated with basal and luminal primary cells.
Basal-derived organoids express higher levels of the basal marker cytokeratin 5, whereas luminal-derived organoids express higher levels of the luminal marker cytokeratin 8. Both basal and luminal markers were detected in basal and luminal-derived organoids in the bulk population, perhaps suggestive of differentiation. Basal-derived organoids contain multilayered epithelium, with outer layers expressing high levels of the basal marker p63, and inner layers having non-detectable levels.
Outer layers also express moderate levels of the luminal marker cytokeratin 8, and inner layers have high levels. While all cells in single-layer luminal-derived organoids stain positively for cytokeratin 8, only select cells contained nuclear p63. Care must be taken when handling matrix gel to ensure that it doesn't harden prior to plating cells into the organoid culture.
DAPI used for microscopy can cause skin irritation. UV light can also be harmful. Adequate personal protective equipment is essential.
We can collect RNA from organoids to perform RNA sequencing, which will tell us about how gene expression profiles change during organoid formation or in response to manipulation. This model has enabled the prostate field to have a reproducible ex vivo assay to study fundamental aspects of epithelial biology and identify regulators in development and differentiation.
