This method can help in the start of genetically-engineered mouse models of prostate cancer and investigation of prostate cancer mechanism through in vivo and in vitro analysis. The main advantage of this technique is that it allows for a complete analysis of the prostate cancer mouse model, all the way from tissue dissection to cell isolation and culturing. After exposing the urogenital system, or UGS, firmly grasp the urinary bladder with medium blunt forceps, and lift the entire system up from the mouse abdomen.
Slide a pair of scissors under the bladder and prostate all the way to the spine to make an incision through the spinal cord, and cut through any remaining connections to the abdominal cavity to allow removal of the entire UGS. Transfer the UGS to a six-centimeter Petri dish containing two to six milliliters of PBS under a dissecting microscope. And use a pair of fine forceps and microdissection scissors to carefully clear all of the fat from both the dorsal and ventral sides of the tissue system without snipping any prostate tissue.
When all of the fat has been removed, pull the bladder with the forceps and excise the bladder at its base. Place the remaining tissue ventral side up and holding one duct ends with the forceps, follow the vessel to its base with scissors and excise the vessel at its base. Remove the same vessel on the other side and insert the forceps between the seminal vesicles and prostate to allow snipping of any adjoining connective tissue as necessary.
Then trace the seminal vesicles to their base at the urethra and remove the vessels without puncturing them. When both vesicles have been removed, place the tissue dorsal side up so the dorsal lobes which resemble a butterfly's wings are visible. Holding each dorsal lobe with forceps, cut the tissue at its base with scissors to collect the dorsal lobes and turn the tissue to the ventral side.
Collect the lateral lobes, which are small and usually wrap the urethra on the side and are wedged between the anterior, ventral, and dorsal lobes in the same manner. Collect the ventral lobes, which are larger than the lateral lobes and lie on the urethra ventrally. Then cut and discard the urethra to harvest the anterior lobes.
To process the prostate tissue for 3D culture, transfer the lobes to a 10-centimeter dish containing two to three milliliters of DMEM, and use a scalpel to mince the prostate tubules as finely and evenly as possible. Transfer the tissue fragments to a sterile tissue culture hood and add the tissue solution to a new 15-milliliter tube. Bring the volume in the tube up to nine milliliters with fresh medium, and add one milliliter of TenX collagenase stock solution to the tissue mixture.
After vortexing, incubate the tube with shaking for two hours at 37 degrees Celsius to degrade the extracellular matrix. At the end of the incubation, collect the tissue by centrifugation and re-suspend the pellet in two milliliter of warm 05%strips in EDTA. After five minutes of 37 degrees Celsius to facilitate cleaving of the cell-to-cell and cell-to-matrix adhesions, use a P1000 pipehead with a wide board tip to triturate the tissue eight to 10 times to break up any clumps.
Repeat the dissociation with a P200 pipehead tip. Then neutralize the trypsin with three milliliters of complete medium. Mix 500 units of DNASE1 into the tissue slurry and pass the solution five to 10 times through a five milliliter syringe equipped with an 18-gauge needle, followed by five times through a 20-gauge needle.
When no more large tissue pieces are visible, filter the suspension through a 40-micrometer filter into a 50-milliliter conical tube, and collect the cells by centrifugation. For cell plating and culture, re-suspend the pellet in 0.5 milliliters of complete prostate epithelial cell growth medium, and allude the cells to a five times 10 to the fifth cells per milliliter of prostate epithelial cell growth medium concentration. Mix the cells with basement membrane extracellular matrix at a two to three volume of solution ratio, and plate the cell suspension in the appropriate experimental cell culture container.
Then place the cells in a cell culture incubator for 30 minutes. When the extracellular matrix has solidified, cover the culture with pre-warmed complete prostate epithelial cell growth medium, taking care not to disturb the basement membrane extracellular matrix plug. Then return the cells to the cell culture incubator for five to 10 days, refreshing half of the medium every two to three days.
To harvest spheres, aspirate the medium carefully without disturbing the basement membrane extracellular matrix gel plug, and add one milliliter of Disbase solution for 100 microliters of basement membrane extracellular matrix. Using a cell scraper, scrape along the bottom of the plate to lift the basement membrane extracellular gels from the bottom of the plate into the supernatant, and pipe up the entire solution one time to break up the plug into smaller pieces. Incubate the extra cellular matrix pieces in the cell culture incubator for one to two hours until the basement membrane extracellular matrix has completely dissolved.
Then transfer the cell suspension to a 15-milliliter tube. And collect the cells by centrifugation for further downstream manipulation. The mouse prostate is composed of four pairs of lobes located dorsally and ventrally to the seminal vesicles and urethra.
The prostate lobes are composed of multiple gland profiles, each comprised of a lumen, surrounded by secretory epithelial cells. The shapes of the lobes, the organization of the cells and the nature of the secretion vary from lobe to lobe. The isolated prostate cells begin growing into organoids as early as four days after basement membrane extracellular matrix culture.
Over the next one to six days, most of the cells grow into solid spheres with a fraction of the spheres exhibiting a partial or full lumen. The spheroids also demonstrate a strong beta catenin staining at the cell-to-cell junctions that co-localize with F-actin staining. While attempting this procedure, it's very important to follow meticulously the microdissection steps.
For example, isolate the prostate lobes while they're still attached to the urethra, so you know which lobe is which based on where they are with respect to the urethra. Following the dissection procedure, the isolated lobes can be used for downstream analysis, such as RMA sequencing, Western blotting or immunostaining. The primary 3D culture technique allows you to gain further insights into the prostate cancer mechanism, because you can monitor life with spheroids for morphology and behavior changes and identify any neoplastic changes in the same.
In summary, this prostate dissection and culture methods can be incorporating to various downstream applications to provide valuable information for investigating the mechanism of prostate cancer using genetically engineered mouse models.
