An Optimized Method To Isolate Female Mouse Urethral Epithelium For Single-Cell And Immunofluorescence Analysis

Our group is interested in studying epithelial patterning in the genitourinary tract. This protocol represents an optimized method to isolate the epithelial lining of the female mouse urethra for downstream molecular and cellular analysis. The methods described in this paper will aid in research on the female urethral lining and its role in immune defense.

To begin, place the euthanized female CD1 mouse 6 to 12 weeks of age in a supine position on the dissection table. Spray 70%ethanol on the abdomen to wet the hair. Using forceps, lift the skin on the abdomen.

Then with small scissors, make a vertical incision toward the lower body, ending at the external urethral opening. Identify the bladder and gently lift it with forceps, ensuring it is empty and free of urine. Using scissors, trim the fat and connective tissue around the bladder to expose the butterfly-shaped structure where the two pubic bones join at the pubic symphysis.

Observe the urethra running beneath the pubic symphysis. Then lift the pubic symphysis with forceps and cut it open using scissors. Remove the pubic bone fragments to expose the thin urethra lying on the ventral surface of the vaginal wall.

Using fine forceps, gently pull the bladder upward from the body cavity. Run scissors between the urethra and the vaginal wall to separate the urinary and reproductive tracts. Maintain slight tension on the bladder while cutting along the back of the urethra to separate it from the vaginal wall.

Continue cutting to the distal urethral opening to obtain the full length mouse urethra, measuring 1.2 to 1.5 centimeters in length. Next, separate the urethra from the bladder by making a single cut at the bladder neck. Pool four to five urethras in a sterile Petri dish containing fresh HBSS free of calcium and magnesium.

Under a dissecting microscope, trim the fat and connective tissue surrounding the urethra using fine point forceps and scissors. Then with scissors, cut each urethra crosswise into two equal sized pieces. Place the tissue pieces on top of a 100 micron nylon mesh strainer.

Wash the tissue pieces with one milliliter of HBSS, then transfer them into a 1.5 milliliter microcentrifuge tube containing one milliliter of 1%trypsin prepared in HBSS supplemented with antibiotics and antimicotics. Next, incubate the sample on a rotator at 30 revolutions per minute and four degrees Celsius for 15 to 30 minutes. After digestion, add 10 millimolar magnesium chloride prepared in HBSS to the tube, followed by 10 micrograms per milliliter of DNAse I prepared in PBS.

Incubate the tube for 15 to 30 minutes on a rotator placed inside a 37 degrees Celsius hybridization oven. To quench the trypsin, transfer the urethral tissues into a 1.5 milliliter microcentrifuge tube containing 10%sheep serum in HBSS at room temperature. After five minutes, transfer the urethral tissues into a new tube containing ice cold HBSS, and then into a sterile Petri dish containing ice cold HBSS.

While viewing the sample under a microscope, hold one end of the urethral piece with a pair of forceps. Using another pair of forceps, pass along the entire length of the urethral piece to extrude the transparent epithelial sheet. Process some of the isolated urethral epithelial sheets directly for whole mount immunostaining to visualize epithelial cells and epithelial-associated macrophages in situ.

Use a small plastic pipette to transfer the epithelial pieces in a drop of HBSS isolated from four to five pooled mouse urethras into a sterile Petri dish. Use scissors to mince the epithelium into small fragments. Transfer the minced epithelial fragments into a two milliliter microcentrifuge tube containing 1.5 milliliters of pre-warmed Accutase digestion solution at 37 degrees Celsius.

Incubate the sample on a rotator at 37 degrees for one hour. After incubation, transfer the entire contents into a five milliliter centrifuge tube containing two milliliters of wash buffer composed of calcium and magnesium-free 1X PBS and 1%BSA. Centrifuge the suspension at 600 G for five minutes at four degrees Celsius using a swinging bucket rotor.

Next, aspirate the supernatant using a gentle vacuum. Then use a one milliliter filter tip pipette to resuspend the pellet in one milliliter of wash buffer. Break up the pellet by pipetting up and down until the clumps become smaller.

Pass the suspension through 23.5 gauge and 26.5 gauge needles, each attached to a one milliliter syringe using four strokes for each needle. Next, add one milliliter of wash buffer to wet the surface of a 30 micron pre-separation filter placed over a five milliliter tube. Using the syringe connected to the 26.5 gauge needle, transfer one milliliter of the cell suspension through the filter.

Then wash the tube with one milliliter of wash buffer and pass it through the filter. Pellet the cells by centrifugation at 600 G for five minutes at four degrees Celsius using a swinging bucket rotor. Aspirate the supernatant.

Resuspend the pellet in 100 microliters of wash buffer and add 100 microliters of red blood cell lysis buffer. After exactly three minutes, add four milliliters of wash buffer and mix. Harvest the cells by centrifugation at 600 G for five minutes at four degrees Celsius using a swinging bucket rotor.

Remove the supernatant prior to resuspending the pellet in 50 to 200 microliters of wash buffer depending on the pellet size. Mix a five microliter aliquot of cells with five microliters of trypan blue and load the mixture onto a hemocytometer to assess cell yield and viability. Assess the cell viability and quantify distinct cell populations using flow cytometry.

Culture urethral epithelial single cells in a basement membrane matrix and organoid expansion media to generate three-dimensional urethral epithelial organoids. Perform hematoxylin eosin staining and fluorescent immunostaining on embedded organoid sections to visualize organoid structure and cellular composition. The isolated urethral epithelial sheaths had a higher transparency than the stromal tissue after separation.

Whole mount immunostaining of the epithelial sheaths showed strong labeling with the E-cadherin CDH1 antibody. Labeling the sheets with the antibody to F480 showed the macrophages forming branching dendritic structures between the epithelial cells of the urethra. Single cells isolated from the urethral epithelium showed consistently high viability by trypan blue exclusion on a hemocytometer and flow cytometry using the amine reactive Ghost Dye V 450.

The isolated cells contained an enriched epithelial cell population after gating on the live cells. Approximately 1.3%of the CD45 positive immune cells and 1.18%macrophages were observed in the single cell suspensions. Single epithelial cells isolated from the urethra remained viable and formed organoids in a three-dimensional matrix with organoid growth observed up to day six in culture.

Hematoxylin eosin staining showed two distinct morphologies of the urethral epithelial organoids, including large hollow organoids and smaller solid organoids. Both the hollow and the solid urethral epithelial organoids were stratified and expressed KRT5 and P63 in the basal layer. They expressed KRT-13 in the suprabasal and intermediate cell layers.

The organoids generated from the proximal urethra showed a higher proportion of hollow organoids while the organoids from the distal urethra formed more solid spheres. This protocol allows researchers to study urethral cell types and self-renewable capacity of epithelial cells from the proximal and distal urethra. One of the most important considerations of this protocol is the careful dissection of full-length urethra and optimal enzymatic digestion to obtain viable urethral epithelial cells for downstream analysis.

The epithelial cells produced by this protocol can be used for multiple applications, including single-cell RNA sequencing, bulk RNA sequencing, flow cytometry, immunostaining, and organoid culture.