Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

This protocol is significant because it allows the generation of testicle organoids, which can be used as a platform for studying testicle morphogenesis and for high tropo drug and toxicity screenings. The main advantage of this technique is that it is highly reproducible, requires a relatively low number of cells and can be used to generate a large number of testicular organoids with testis-specific architecture. This method can be applied to study other systems, such as pluripotent stem cells or pancreatic islet cells with the necessary optimizations.

Demonstrating the testicular tissue enzymatic digestion procedure will be Anna Voigt, a PhD student from my laboratory. Collect the testis into a sterile beaker and wash with PBS containing 1%penicillin streptomycin. After washing transfer the testis to a 100 millimeter tissue culture dish with PBS containing 1%penicillin streptomycin.

Use autoclaved scissors and forceps to remove the tunica vaginalis and epididymis. Transfer the isolated testis to a new 100 millimeter dish and wash thoroughly with PBS containing 1%penicillin streptomycin. Cut the testis along the longitudinal axis directly under the tunica.

Then peel the testis out of the tunica using two forceps and place it into a new 100 millimeter dish containing 1 milliliter of DMEM with 1%penicillin streptomycin. Mince the peeled testis with sterile scissors into one to two millimeter tissue pieces. After chopping use sterile forceps to remove the white fragments of connective tissue.

Transfer the minced tissue pieces into solution A in a 50 milliliter tube and top it up to 50 milliliters with DMEM to obtain a concentration of 0.4 milligrams per milliliter for colagenase 4S and a concentration of 0.8 milligrams per milliliter for colagenase 4W. Place the tube containing the tissue pieces into a 37 degree Celsius water bath for 30 minutes. Gently invert the tube every five minutes.

After 30 minutes, centrifuge the tube at 90 times G with breaks at 25 degrees Celsius for 1.5 minutes. Discard the supernatant, ad 40 milliliters of solution B, and top up to 50 milliliters with DMEM to obtain a concentration of 1.2 milligrams per milliliter of colagenase 4W. Place the tube into a 37 degree Celsius water bath for 30 minutes and gently invert the tube every five minutes.

Centrifuge the tube at 90 times G with breaks at 25 degrees Celsius for 1.5 minutes. After that discard the supernatant, and wash once with PBS with 1%penicillin streptomycin. Carefully collect the tubules from the top and place into a new 50 milliliter tube.

Top up the tube with PBS up to 50 milliliters. Centrifuge the tubules at 90 times G with breaks at 25 degrees Celsius for 1.5 minutes. Discard the supernatant and add fresh PBS to wash two additional times.

After the last PBS wash remove the supernatant and re-suspend the seminiferous tubules in five milliliters of PBS. Then, add 15 milliliters of 0.25%Trypsin EDTA to the tubules. Place the tube in a 37 degree Celsius water bath and gently invert every two minutes.

After five minutes evaluate the enzymatic digestion of tubules to single cells under the microscope with 10 microliters of sample on a tissue culture plate. Every two minutes place the sample under the microscope to observe. If mostly single cells can be detected stop the reaction with five milliliters of FBS and filter through a 70 micron mesh and then through a 40 micron mesh.

Centrifuge the single cells at 500 times G with breaks at 25 degrees Celsius for five minutes. Re-suspend in 50 milliliters of enrichment medium, and count the number of viable cells using a hemocytometer. Transfer 20 million of this starting cell population into each of two ultra-low attachment 100 millimeter Petri dishes and incubate in a tissue-culture incubator at 37 degrees Celsius, 5%carbon dioxide and 21%oxygen for two days.

Cede 20 to 25 million cells of the remaining starting cell population per 100 millimeter tissue culture dish in a total volume of eight milliliters of enrichment medium. Place the dish into an incubator and after 1.5 hours ensure that the majority of Sertoli cells are attached to the plate. Slightly tilt two plates to collect and combine the supernatants into one new 100 millimeter plate and place it back into the incubator.

After one hour, again combine the supernatants of two plates to a new 100 millimeter plate. Place the plates back into the incubator overnight. Then collect the enriched germ cells in two fractions, non-adherent fraction and slightly adherent fraction.

Collect the supernatant as the non-adherent fraction. For the slightly adherent fraction wash the plates gently with PBS and treat with two milliliters of a one to 20 dilution of 0.25%Trypsin EDTA for five minutes at room temperature. Stop the reaction by adding two milliliters of enrichment medium and collect in the non-adherent fraction tube.

Combine the starting cell preparation and the enriched germ cells in a tube to obtain a working cell preparation containing 25%germ cells. Centrifuge at 500 times G with breaks at 25 degrees Celsius for five minutes. Discard the supernatant and re-suspend the cells in 20 milliliters of organoid formation medium to reach a concentration of 2.4 million cells per milliliter.

In a new tube add one milliliter of the solution containing cells. Add 0.5 milliliters of surfactant rinsing solution to each well in a micro well plate. Ensure that no air bubbles are trapped in the well.

To remove air bubbles centrifuge the plate at 2, 000 times G with breaks at 25 degrees Celsius for two minutes. Observe the plate under a low magnification inverted microscope to verify that the bubbles have been removed from the micro wells. Cover the plate with a lid and incubate for 30 minutes at room temperature.

After the treatment is complete remove the rinsing solution and immediately wash the plate with sterile water or PBS. Add 0.5 milliliters of organoid formation medium to each well and centrifuge at 2, 000 times G with breaks at 25 degrees Celsius for two minutes to remove any trapped air bubbles. Observe the well under a low magnification inverted microscope to verify that the air bubbles have been removed.

Add 0.5 milliliters of the working cell suspension and gently mix by pipetting up and down. Centrifuge at 500 times G with breaks at 25 degrees Celsius for five minutes. Use an inverted microscope to verify that the cells have clustered within the micro wells.

Transfer the plate into a cell culture incubator and culture for five days. Change half of the medium every other day. To recover the organoids use a wide-mouth pipette to gently pipette the medium up and down.

This allows the organoids to come out of the micro wells. Collect the organoids, fix and perform immuno cytochemistry for germ cell marker, Sertoli cell marker, peritubular myoid cell marker and Leydig cell marker and visualize under a confocal microscope. In this study isolated cells from one-week old porcine testis that were cultured in micro wells self-organized into spheroids with delineated and distinct exterior and interior compartments.

The two compartments were separated by a collagen IV positive basement membrane. GATA4 positive Sertoli cells and UCHL1 positive germ cells were in the exterior compartment on the basement membrane. aSMA positive peri-tubular myoid cells were localized along the inside of the basement membrane while CYP450 positive Leydig cells were in the center of the interstitium.

This structure is similar to In Situ conditions, where Leydig cells, peri-tubular myoid cells, are located in the interstitium, in the interstitial compartment and germ cells, Sertoli cells are located at the seminiferous epithelium. It is important to ensure that cells being used to generate organoids have optimum quality. Careful attention should be paid during the trypsinization stage of the enzymatic digestion.

Following this procedure the organoids can be cultured long-term to study germ-cell differentiation or analyze for gene expression and secreted proteins or hormones. The ability to create organoids with testis-specific cell associations provides a novel in-vitro system to study cell-cell interactions important for testis development and germ cell function.