Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

These protocols enable the user to produce a large number of structurally mimetic and endocrine functional testicular organoids in both ECM and DCM-free environments in 2D or 3D culture conditions. These techniques are highly reproducible, easily applied to standard biological laboratory settings, and only utilize common tools and materials. Testicular organoids are a novel tool for simulating spermatogenesis and reproductive endocrinology in vitro, and might one day enable the study of in vitro gametogenesis.

Begin by placing the euthanized mouse supine on a dissection mat and sterilizing the abdomen with 70%ethanol. Tent the skin of the lower abdomen with forceps and open it with scissors. Locate the testes in the lower left and right inguinal regions of the abdomen and cut their connections to the vas deferens and any anchoring connective tissue.

Then lift the entire testes from the animal. Place it in a Petri dish of pre-equilibrated BM.Under a dissection microscope, make a small incision in the tunica albuginea on one end of each testis with either small micro dissection scissors or by gently tearing with two fine forceps. While holding the testis from the opposite end of the incision, gently squeeze it with fine forceps and push in a sweeping motion towards the hole in the tunica, which will release the testicular tissue as one cohesive piece.

Cut the tissue into smaller pieces. Place the pieces into one milliliter of prewarm dissociation solution one and incubate them at 37 degrees Celsius for 10 minutes. After the incubation, gently triturate the testes pieces 50 times with a P1000 pipette.

Ensure that the tubules separate from one another and from the interstitial tissue. If clumps remain, incubate for an additional five minutes and triturate again. Add 33 microliters of hyaluronidase stock solution per one milliliter of the dissociation mixture.

Then triturate 50 times with a P1000 pipette and incubate the sample at 37 degrees Celsius for five minutes. After the incubation, triturate it 50 times with a P200 pipette. After ensuring that no visible tubules or clumps of cells are present, quench the dissociation enzymes by adding FBS to 10%of the total volume of the sample and triturate it several times with a P200 pipette.

Wet the center of a 40 micrometer cell strainer with media and aspirate it away, then add the sample to the pre-wetted strainer and filter it to produce a single-cell suspension. Centrifuge the suspension at 100 times g for seven minutes. Discard the supernatant and resuspend the cells in fresh BM.Count the viable cells on a hemocytometer using trypan blue exclusion, then re-centrifuge and resuspend them in fresh BM.For 2D ECM-free culture, plate single-cell suspensions directly onto four-well chamber slides and place the slides into a 35 degree Celsius incubator.

For 2D ECM-culture, dispense 100 microliters of cold one-to-one diluted extracellular matrix medium into a four-well chamber slide, ensuring that the gel covers the dish bottom. Place the slide in the incubator for a minimum of 30 minutes to allow the ECM to polymerize into a gel, then add the cell suspension on top. For a 3D ECM-free culture, place agarose 3D Petri dishes into a 24-well culture dish and cover them with one milliliter of BM.Let the dishes equilibrate in BM for at least 30 minutes in a 35 degree Celsius incubator, then discard the BM and repeat the equilibration once more with one milliliter of fresh BM.Remove all BM from the well and dispense 200 microliters of fresh BM around but not inside the center recess of the 3D Petri dish, then collect any remaining BM from inside the center cell seeding recess of the micro well insert.

Dispense the single-cell suspension into the center recess and gently triturate up and down. Place the dish into a humidified 35 degree Celsius incubator for culture. On the next day, slowly and carefully remove existing media from around the agarose 3D Petri dish and replace with one milliliter of fresh BM.The organoids should have compacted overnight, allowing them to rest at the bottom.

For 3D ECM culture, prepare a single-cell suspension by combining equal parts of the cell suspension in BM with cold pre-thawed ECM. Triturate several times to ensure it is well mixed. Then immediately dispense the mixture into a four-well chamber slide, ensuring that it covers the entire bottom of the plate.

Incubate the chamber slides at 35 degrees Celsius and allow its contents to polymerize for at least 30 minutes. After the polymerization, add 500 microliters of BM on top of the culture. Culture all organoid model types at 35 degrees Celsius.

For all culture types, exchange half of their media with fresh BM every two days. This protocol was used to achieve organoid generation within 24 hours using juvenile murine testicular cells. Successfully generated tissues were initially observed as 3D cell clusters.

In ECM environments, the cell clusters possessed clear margins between the cluster and their surrounding environment. Cell clusters were also observed to migrate across the ECM and fuse together. The 3D ECM culture required significantly more time to assemble de novo structures than all other culture methods.

And the 3D ECM-free culture produced the largest clusters with a single large and compact cluster within each well of the 3D agarose Petri dish. To assess whether the organoid models resemble the native tissue, the biological constructs were probed for cell-specific markers and visualized with immunofluorescence. The 2D ECM and 3D ECM-free methods produced organoids with an inner morphology highly mimetic of testicular compartmentalization.

A long-term analysis was performed on the 3D ECM-free assembled organoids. The organoids were cultured for 14 days and then probed for cell and structure-specific markers. Tubule-like structures were observed.

The 3D ECM-free organoids were also studied for endocrine function in a 12-week culture with gonadotropin stimulation. Testosterone and inhibin B were both quantified from organoid-conditioned medium. After 12 weeks, gonadotropins were removed for 48 hours, causing testosterone and inhibin B concentrations to decrease.

When the gonadotropins were returned, both hormone concentrations significantly increased, demonstrating proper endocrine responsiveness. Organoid assembly takes advantage of the autonomous behavior of viable immature testicular cells. Creative experiments can be easily devised through using customized cell sorted or hybrid cell mixtures.

Following cell seeding, live video imaging or time-lapse imaging can be used to quantify organoid self-assembly. Across culture, organoids and condition medium can be collected for histological and amino assay analyses.