Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina

Research into disorders that primarily affect people of female gender is understudied and underfunded. Pelvic organ prolapse is the disorder strongly associated with female gender. It occurs when muscles and ligaments weaken and cause pelvic organs to drop lower in the pelvis, creating a bulge in the vagina.

Little is known about cellular interactions in the context of this pathophysiology, nor how tissue level factors impact success of surgical interventions. Isolation of primary fibroblasts from human vagina may be useful for studying biological mechanisms underlying pelvic organ prolapse. Pelvic organ prolapse commonly affects older or postmenopausal individuals.

Isolation and proliferation of primary fibroblasts study of this condition is challenging due to reduced cell populations and clonogenic ability of older donors. Most papers using human vaginal samples utilize tissue from premenopausal individuals with pelvic organ prolapse. Although a few papers reported use of samples from both premenopausal and postmenopausal individuals, they did not describe in enough detail the protocol used to successfully isolate fibroblast from older or postmenopausal donors.

Isolation and disease modeling using fibroblasts from postmenopausal tissue may be essential to understanding cellular pathophysiology of pelvic organ prolapse as this condition has the highest prevalence in individuals in the decade following menopause, We describe a method for isolation and culture of a fibroblast-enriched single cell suspension from human vaginal tissue using a combination of mechanical and enzymatic dissociation. This article describes a reliable protocol on how to acquire postmenopausal or aging human vaginal fibroblasts. Harvested tissues were analyzed histologically to validate the protocol.

Measure and cut the tissue to the size of approximately one centimeter squared. Ensure precise measurement of the biopsy size. Prepare two to three additional vaginal biopsies, measuring one centimeter squared each from a single donor and set the biopsies aside.

After placing the vaginal biopsy in a cell culture Petri dish, mince the vaginal tissue into small fragments, using two sterile scalpels. Ensure the tips of the blades are perpendicular to the surface of the tissue. Apply equal and steady pressure onto the tissue surface, using two scalpels.

Perform an alternating pulling action to cut the tissues into very small pieces. Repeat this mincing technique, using two scalpels, until the sample is of uniform consistency with one to two millimeter pieces. Add two to three milliliters of serum-free cell culture media to the plate, suspending the minced tissue.

Pipette the solution containing tissue fragments up and down to break up any clumps. Transfer the solution containing tissue fragments into a 15 milliliter conical tube. Add additional serum-free cell culture media until the total volume is 10 milliliters.

Store Liberase in 230 microliter aliquots for use. Add 230 microliters of Liberace to the tube. Incubate the tubes for three hours at 37 degrees Celsius with constant vigorous agitation, using a sample mixer.

Maintain constant agitation. Vortex the tubes at 30 minute intervals during the incubation. Centrifuge samples for five minutes At 3000 G.Remove and discard the supernatant.

Re-suspend the pellet in one to two milliliters of cell culture media with 10%fetal bovine serum to dilute the Liberase. Vigorously pipette until the pellet is fully re-suspended. Strain the tissue enzyme suspension by gently pipetting the solution over the cell strainer.

Pull cell suspensions from multiple tissue biopsies of the same donor together. With the plunger of a sterile five milliliter syringe, press through the tissue enzyme suspension over the strainer. Repeat this step until the remaining tissue fragments appear to be fully pressed through.

Plate pulled cell suspension from multiple vaginal biopsies from the same donor on a single Petri dish. After overnight incubation at 37 degrees Celsius, observe cells with a phase contrast microscope. Ensure focus of the microscope on the bottom of the plate.

A small number of fibroblasts will be attached to the bottom. Here's a diagrammatic representation of the protocol, highlighting key steps. Figure two shows a phase contrast image of suspended cells on day zero.

Figure three is a phase contrast image of vaginal primary fibroblasts on day 14 of culture at 100X magnification from a pulled suspension of three one centimeter squared tissue biopsies. Table one shows the results from using other protocols to attempt vaginal fibroblasts isolation in this current study. Fibroblasts were identified by positive staining of the menton.

Immunofluorescence analysis was performed on premenopausal and postmenopausal vaginal tissues shown here. Images were taken at 200X magnification. Isolated fibroblasts were also stained with alpha smooth muscle actin and F actin in premenopausal and postmenopausal tissue samples.

We developed an optimized and reliable protocol for isolation of primary fibroblasts from vaginal mucosa in older donors. Use of previously published protocols for animal and human tissue for dissociation of primary cells failed to extract any fibroblasts from human vaginal samples in this study. We hypothesize two probable reasons for challenges of cell dissociation from human vaginal tissue.

Dermal thickness of mucosa is greater in older donors and when compared to that of non mucosal tissue, and density fibroblast may be markedly reduced in older individuals. A few critical steps should not be modified. The implementation of a very rigorous mechanical digestion, using the two scalpel technique, and pulling together the cell suspensions of three to four full thickness biopsies of a single donor.

Our protocol has a distinct advantage. The mechanical and enzymatic digestion lead to better yields for postmenopausal tissue, but do not have a negative impact on premenopausal samples. We acknowledge several limitations to our protocol.

Access to human vaginal tissue may be challenging in situations where vaginal prolapse surgery is not being performed, and the need for pulling of cell suspensions of multiple tissue biopsy samples may also be a limitation. This protocol for requiring human vaginal fibroblasts will be a useful tool for future investigations in pelvic floor disorders, especially in post-menopausal individuals, as well as an important addition to women's health research.