Establishment and Maintenance of Patient-derived Prostate Cancer Organoids: A Detailed Experimental Protocol

The research focus on establishing and maintain the patient-derived prostate cancer organoid for personalized treatment studies. Advances include the optimized organoid culture system to augment the fidelity preservation and drug screening for personalized prostate cancer therapy. To begin place, collected tissue blocks into 50 milliliter sterile conical centrifuge tubes and label each tube accordingly.

Add preservation solution to each tube until the tissues are fully submerged. Using sterilized tweezers, transfer the prostate cancer tissue samples into a medium-sized culture dish. Wash the tissues two to three times with two milliliters of DPBS to remove blood and debris.

Then, carefully remove adipose tissue, muscle, and other non-epithelial components using fine-tipped tweezers, keeping only epithelial regions. Using scissors, mince the cleaned tissue into small fragments measuring approximately one to three cubic millimeters. Now, transfer the tissue fragments into tissue processing tubes and add tumor tissue digestion solution to each tube.

Start the digestion device and run the medium-hard tissue dissociation program for enzymatic mechanical dissociation. After that, add an equal or double volume of FBS-supplemented adDMEM and F12 medium to the cell suspension to terminate enzymatic digestion. Pre-wet a 100 micrometer cell strainer with one milliliter of adDMEM/F12 medium and filter the cell suspension through it.

Then, rinse the tissue processing tube and cell strainer twice with five milliliters of adDMEM/F12 medium supplemented with ROCK inhibitor solution. Collect the filtrate into a 50 milliliter sterile conical centrifuge tube. Pass the collected suspension through a 70 micrometer strainer and transfer all filtered suspension into a new 50 milliliter tube.

Then, centrifuge the collected cell suspension at 200 G for five minutes at 25 degrees Celsius. Carefully discard the supernatant and resuspend the cell pellet in two milliliters of red blood cell lysis buffer. Then, incubate the suspension on ice for two to three minutes.

To terminate the lysis, add six milliliters of adDMEM/F12 medium supplemented with ROCK inhibitor. Transfer the mixture to a 15 milliliter sterile conical centrifuge tube and centrifuge at 200 G for five minutes at 25 degrees Celsius. After discarding the supernatant, wash the cell pellet one to two times with the same supplemented medium and transfer the final suspension into a 1.5 milliliter Eppendorf tube.

Now, mix 10 microliters of the cell suspension with 10 microliters of trypan blue solution. Resuspend the cell pellet in one milliliter of adDMEM/F12 medium. Pipette six microliters of the diluted suspension onto a hemo cytometer and count the cells under a microscope.

Now, centrifuge the cell suspension at 200 G for five minutes at 25 degrees Celsius. Transfer the final cell pellet into a 1.5 milliliter Eppendorf tube. Determine the required volume of matrix gel from the cell count.

Using pre-chilled pipette tips, carefully add the matrix gel to the cell pellet. Mix the cell gel mixture gently on ice to avoid air bubbles and maintain matrix gel integrity. Now, dispense approximately 30 microliters of the cell matrix gel mixture per drop into a low adhesion, 24-well culture plate.

Place the culture plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for five minutes to allow preliminary gel solidification. Invert the plate and let it sit for 20 minutes to retain the three-dimensional form of the gel. Add 800 to 1, 000 microliters of culture medium to each well.

Using a pre-chilled pipette tip, mechanically disrupt the matrix gel in each well to fragment the organoid clusters. Rinse each well one to two times with adDMEM/F12 medium supplemented with ROCK inhibitor solution. Transfer the fragmented clusters and culture medium into a 15 milliliter sterile conical centrifuge tube.

Centrifuge the tube at 200 G for five minutes at 25 degrees Celsius. After discarding the supernatant, resuspend the resulting pellet in two milliliters of pre-warmed recombinant trypsin-like enzymes solution. Pipette the mixture up and down to begin dissociation.

Then, incubate the tube at 37 degrees Celsius for 15 to 30 minutes. Now, add an equal or double volume of adDMEM/F12 medium supplemented with FBS to stop the enzymatic reaction. Centrifuge the suspension at 200 G for five minutes at 25 degrees Celsius.

Then, discard the supernatant. To begin matrix gel removal, resuspend the pellet in two milliliters of cell recovery solution. Incubate the suspension at four degrees Celsius for 20 to 40 minutes to dissolve the residual matrix gel.

Centrifuge the sample at 200 G for five minutes at four degrees Celsius. After discarding the supernatant, wash the pellet two times using adDMEM and F12 medium to eliminate any remaining matrix components. Finally, transfer the pellet to a 1.5 milliliter sterile Eppendorf tube for further processing.

Seed the dissociated organoid clusters following the reseeding procedures. Initial organoid formation was observed between day five and day seven of culture with bright-field images showing compact and well-defined spheroids or glandular-like structures. Hematoxylin and eosin staining revealed preserved glandular architecture in organoids derived from well-differentiated adenocarcinoma, resembling the matched patient tissue.

Immunohistochemistry showed strong nuclear androgen receptor expression in both organoids and primary tumors derived from hormone-sensitive prostate cancer tissues. CK5, PSMA, and NKX3.1 were robustly expressed in both organoid and tumor samples, supporting epithelial origin and lineage fidelity. KI-67 staining showed that the organoids retained a proliferative index comparable to the original tumor tissues.

We have developed a robust protocols for maintaining patient-derived prostate cancer organoids, enabling personalized drug testing and biomarker discovery. Our findings provide a reliable platform for studying prostate cancer biology, facilitating personalized therapies and precision medicine advancements. Future research will explore organoid heterogeneity, tumor microenvironment interactions, and drug resistance mechanisms in prostate cancer.