Place harvested prostates in 15mL falcon tube containing cold PBS. Transport ... (10X) with 9 parts F-12/DMEM (3:1) supplemented with 10 mM Hepes and 5%.
Mouse Prostate Epithelial Cell Isolation Protocol Henry Withers 2013 – Roswell Park Cancer Institute Adapted from StemCell Technologies ProstaCult Medium Protocol and David L. Hudson’s “Prostate Epithelial Cell Isolation and Culture” (Methods in Molecular Biology, vol 188) *Note: Avoid the use of glass pipettes and tubes due to the propensity of epithelial cells to stick to these surfaces. Use plastic when possible… Mouse Prostate Isolation: 1.) Sacrifice mouse, secure mouse to styrofoam cooler top, ventral-side up with pins in each limb. Sterilize incision area with liberal amount of 70% EtOH. 2.) Using sterile instruments, grasp the skin ~2cm anterior to the urogenital opening with forceps and make a small cut with scissors close to forceps’ grip at the top of “tented” skin. Using the scissors and blunt dissection technique, make larger cut from original opening in skin and separate skin from underlying muscle. 3.) Carefully cut into muscle layer making sure not to disturb organs (i.e. intestine/colon) found underneath. 4.) Locate the bladder and search for the white, “hooked/curled”, and paired seminal vesicles resting dorsally. The may be hidden up under the intestines/stomach. 5.) Uncurl the seminal vesicle to find the small brown colored anterior prostate (red boxes in figure below) located near the base of S.V.
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6.) Carefully pull apart and cut the brown organ from the white seminal vesicle. 7.) Place harvested prostates in 15mL falcon tube containing cold PBS. Transport on ice. 8.) Under cell culture hood, place prostate tissue in petrie dish and use two sterile scalpels to finely mince tissue. Dissociation: 1.) Transfer minced tissue to 15mL falcon tube with cold PBS and centrifuge at 800 rpm for 5 min. 2.) Remove supernatant and resuspend tissue in 1 part Collagenase/Hyaluronidase (10X) with 9 parts F-12/DMEM (3:1) supplemented with 10 mM Hepes and 5% FBS final concentration. ~3 - 5 mL are needed for 2-3 mouse prostates. 3.) Incubate in a 15ml falcon tube at 37◦C for at least 3 hours with shaking. 4.) To separate epithelial organoids from the digested stromal components, centrifuge for 20 seconds at 170g and then carefully discard supernatant. Resuspend the pellet with 10 mL PBS and repeat this procedure two more times. **It is possible to resuspend cells in appropriate media and plate them after this step; however, you will likely have a mixture of organoids (clumps of epithelial cells) and single cells. To obtain a single cell suspension with the greatest removal of fibroblasts, continue with the following steps: 5.) Resuspend the pellet in 5 – 6 mL of 0.25% Trypsin-EDTA and leave on ice for 1 hour. 6.) While the cells sit in Trypsin, prepare: a. Modified Hanks’ Balanced Salt Solution (HBSS): HBSS with 15 mM Hepes and 2% FBS final concentration (PBS is an acceptable substitute for HBSS). b. Thaw dispase and DNase I. 7.) After 1 hour, add 10 mL of cold HBSS modified (15 mM Hepes, 2% FBS) and centrifuge at 350 x g for 5 minutes with the brake on. 8.) Remove as much of the supernatant as possible. 9.) Add 2 mL of pre-warmed 5 mg/ml Dispase and 200 µL DNase I (1 mg/ml). Pipette the sample up and down with P-1000 pipette for 1 minute. 10.) Add 10 mL of cold HBSS modified (15 mM Hepes, 2% FBS) and then filter the cell suspension through a 40 µm cell strainer into a new 50 mL falcon tube. 11.) Centrifuge at 350 x g for 5 min with the brake on. Discard the supernatant. 12.) Resuspend the cells in desired culture media. 13.) 4 anterior prostate lobes from two mice should yield enough cells to be plated in one or two 100mm tissue culture dishes.
