Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina

Summary

This protocol demonstrates a reliable and effective technique to isolate primary fibroblasts from either premenopausal or postmenopausal human vaginal tissue. Existing protocols for vaginal fibroblast isolation do not consider the challenges of cell isolation from senescent tissue. Vaginal tissue was obtained from women after pelvic organ prolapse surgery.

Abstract

Pelvic organ prolapse is a disorder that seriously impacts the quality of life of women. It occurs when muscles and ligaments weaken and cause pelvic organs to drop lower in the pelvis, creating a bulge in the vagina. Surgery to correct pelvic organ prolapse has been a mainstay treatment. Recently, there has been growing interest in studying the tissue composition of patients with prolapse at the cellular level.

There is currently little consensus regarding the effect of donor or patient age on cell-based therapies. Current published protocols for vaginal fibroblast isolation either concentrate on premenopausal tissue or neglect to comment on the age of donor tissue. Most existing protocols use animal models. The consistency of human vaginal tissue is denser than the tissues used in most protocols. In this study, human vaginal tissue was obtained primarily from older donors, which likely contributed to the failure of existing protocols.

The aim of this study is to describe a standard protocol for reliably acquiring human vaginal fibroblasts, regardless of donor age and menopausal status. Results were reproduced using tissue from nine separate donors who underwent pelvic organ prolapse surgery. Six patients were postmenopausal, with the oldest donor being 78 years old. The median age of the tissue donors was 59.

Here, we describe a reliable method for generating a fibroblast-enriched single-cell suspension using a combination of enzymatic and mechanical dissociation and cell suspension pooling of multiple vaginal biopsies from a single donor. Reliable isolation of human vaginal primary fibroblasts may be useful in the study of pelvic organ prolapse as well as microbiome-host interactions.

Introduction

Gender disparity in science and research is an ongoing issue. Research into disorders that primarily affect people of female gender is underfunded¹. Pelvic organ prolapse is a disorder strongly associated with the female gender. It occurs when muscles and ligaments weaken and cause pelvic organs to drop lower in the pelvis, creating a bulge in the vagina². Little is known about cellular interactions in the context of this pathophysiology, nor how tissue-level factors impact the success of surgical interventions³.

Primary cells rather than cell lines are widely recognized as essential for translational clinical research in providing more physiologic relevance⁴. Primary cells are taken directly from the body tissue of interest and may more precisely mimic the in vivo physiology. Isolation of primary fibroblasts from the human vagina may be useful for studying biological mechanisms of pelvic organ prolapse and disease modeling, considering key donor characteristics, such as age and menopausal status.

Pelvic organ prolapse commonly affects older or postmenopausal individuals. The isolation and proliferation of primary fibroblast cells in studying this condition are challenging due to reduced cell populations and clonogenic ability from older donors⁵. In our experience, the use of previously described protocols for the dissociation of vaginal tissue failed to extract any fibroblasts from a standard 1 cm² tissue biopsy.

Through a literature review, we found that similar studies were subdivided into two groups: animal models, such as murine⁶, and human models^7,8. When following the mouse protocol, the use of scissors for tissue processing of human vaginal tissue yielded inadequate mechanical digestion. Extrapolation of protocols using murine tissue and other tissue sites⁹ was unsuccessful.

Most papers using human vaginal samples utilized tissue from premenopausal individuals with pelvic organ prolapse^7,10. Although a few papers reported the use of samples from both premenopausal and postmenopausal individuals¹¹, they did not describe in enough detail the protocol used to successfully isolate fibroblasts from older or postmenopausal donors. Isolation and disease modeling using fibroblasts from postmenopausal tissue may be essential to understanding the cellular pathophysiology of pelvic organ prolapse, as this condition has the highest prevalence in individuals in the decade following menopause¹².

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. Isolated cells were confirmed to be fibroblasts through morphologic examination using phase contrast microscopy and immunofluorescence (IF) to assess the expression of vimentin, F-actin, and α-smooth muscle actin (α-SMA).

Protocol

Vaginal tissue was obtained from women undergoing pelvic organ prolapse surgery. The vaginal tissue collected after the surgery was considered waste material and would otherwise be discarded. The study was performed in compliance with the Institutional guidelines and was approved by the Institutional Review Board of Massachusetts General Brigham.

1. Vaginal tissue harvest from the patient

1. Diagnose pelvic organ prolapse by taking a history and using pelvic exam findings: Pelvic Organ Prolapse Quantification (POP-Q) measurements were obtained in the clinic, showing evidence of pelvic organ prolapse.
2. Use the following inclusion criteria: over 18 years of age; history of symptomatic pelvic organ prolapse; electing for surgical correction of pelvic organ prolapse.
3. Exclude the following: Pregnant women and patients refusing to donate tissue.
4. Trim the excess vaginal tissue as a necessary step of pelvic organ prolapse surgery. This vaginal epithelium is excised in total thickness after the following surgeries: anterior colporrhaphy, posterior colporrhaphy, and colpocleisis.
5. Transport tissue to the lab in a sterile specimen collection cup with normal saline or serum-free DMEM/F12 (Gibco) media. Keep on ice.

2. Tissue processing and digestion

1. Tissue preparation
   1. Measure and cut the tissue to encompass the full thickness of the vaginal epithelium, creating segments of approximately 1 cm².
   2. Ensure precise measurement of the biopsy size 1 cm².
      NOTE: Overestimating the tissue biopsy size may impair tissue digestion.
   3. Prepare 2-3 additional vaginal biopsies measuring 1 cm² each from a single donor and set the biopsies aside.
2. Mechanical digestion
   1. Place the vaginal biopsy in a 10 cm cell culture Petri dish. Pipette 500-1,000 µL of serum-free media supplemented with 100 U/mL penicillin/streptomycin, 2.5 µg/mL amphotericin Bonto the vaginal tissue to keep the tissue from drying out.
   2. Mince the vaginal tissue into small fragments using two sterile scalpels (No. 11 or No. 15) in both hands.
   3. 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 tissue into very small pieces.
   4. Repeat this mincing technique using the two-scalpel technique until the sample is of uniform consistency with 1-2 mm pieces.
      ​NOTE: Mechanical digestion using this two-scalpel technique of a 1 cm² biopsy should take approximately 15-20 min to complete.
   5. Add 2-3 mL of serum-free DMEM/F12 media supplemented with 100 U/mL penicillin/streptomycin, 2.5 µg/mL amphotericin B to the plate, suspending the minced tissue. Pipette the solution containing tissue fragments up and down to break up any clumps.
3. Enzymatic digestion
   1. Transfer the solution containing tissue fragments into a 15 mL conical tube. Add serum-free DMEM/F12 media to the tissue culture dish where vaginal tissue was minced to collect any remaining tissue fragments. Transfer the solution containing tissue fragments to the conical tube. Add additional serum-free DMEM/F12 media until the total volume is 10 mL.
   2. Dissolve 5 mg of Liberase in 1 mL of sterile water (5 mg/mL). Store in 230 µL aliquots at −20 °C for up to a month or −80 °C for 6 months.
   3. Add 230 µL of Liberase (stock 13 U/mL) to the tube, with final concentration 0.3 U/mL.
      NOTE: Liberase activity may vary between batches. Thaw each aliquot immediately before use using a water bath. Avoid repeated freezing and thawing.
   4. Repeat steps 2.1-2.3 for 2-3 additional vaginal biopsies of the same donor.
      ​NOTE: Keep each solution containing tissue fragments from a single biopsy in separate conical tubes. For example, 4 vaginal biopsies in 4 tubes. This is important for optimal cell pelleting after centrifugation.
   5. Incubate the tubes for 3 h at 37 °C with constant, vigorous agitation using a sample mixer.
   6. Place a sample mixer in a CO₂ incubator. Maintain constant agitation. (Sample mixer settings: 25 rpm of rotational motion for 5 s, 30° of reciprocal (tilting rotation) for 5 s, and 2° of vibration (vortexing) motion for 3 s).
   7. During the incubation, vortex the tubes at 30 min intervals.
   8. Centrifuge the samples at 3,000 x g for 5 min. Remove and discard the supernatant.
   9. Resuspend the pellet in 1-2 mLof DMEM/F12 media with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin, 2.5 µg/mL amphotericin B to dilute the Liberase enzyme. Vigorously pipette until the pellet is fully resuspended.

3. Cell culture

1. Removal of non-digested tissue fragments
   1. Place a 100 µm cell strainer over a sterile 50 mL conical container.
   2. Pool the cell suspension from the multiple tissue biopsies of the same donor together.
   3. Strain the tissue/enzyme suspension, pressing through with a 5 mL syringe plunger. Repeat this step until the remaining tissue fragments appear to be fully pressed through.
      NOTE: There may be some mucus from the vagina tissue fragments that is not fully worked through the strainer. Discard the mucus with the cell strainer.
2. Cell culture pooling
   1. Plate pooled cell suspension from multiple vaginal biopsies (from the same donor) on a Petri dish (60 mm x 15 mm).
   2. Incubate the Petri dish in a CO₂ incubator at 37 °C overnight.
   3. Observe and confirm the presence of non-adherent cells with a phase-contrast microscope.
   4. Ensure the focus of the microscope is on the bottom of the plate.
      NOTE: There may be many immune cells in the foreground. A smaller number of fibroblasts will be attached to the bottom of the plate.
   5. Wait for 18-24 h prior to changing cell culture media to ensure adequate cell adherence.
   6. Change the culture medium every three days until 80% confluence occurs. When cells reach 80-90% confluence, expand to a larger flask or freeze aliquots of cells for future use.

Results

Vaginal tissue was collected from 10 independent donors undergoing pelvic organ prolapse surgery. Using the described protocol, cells were isolated from human vaginal tissue. Cell populations had a characteristic elongated, flat, and spindle-shaped appearance. Like other studies, we observed significantly slower cell doubling capacities and reduced clonogenic ability of fibroblasts with increasing age of the donor. These differences were marked when comparing fibroblasts isolated from older individuals (75-78 years old) to those isolated from young individuals (35-47 years old). Cells from middle-aged donors exhibited an intermediate profile (56-61 years old). Cell proliferation rates were initially estimated by direct visualization using a phase-contrast microscope with the same known seeding density.

Figure 1: Diagrammatic representation of the protocol. Please click here to view a larger version of this figure.

Figure 1 shows a diagrammatic representation of the dissociation and isolation protocol. Following these steps, this protocol yielded a 90% success rate of primary fibroblast isolation in nine out of ten donors in sufficient cell numbers to allow for subculturing cells. The median age of donors was 59.

Protocol (Author, year)	Donor tissue of original protocol	Number of Donors	Donor age in years	Fibroblasts obtained	Morphology
Ruiz-Zapata et al., 2013	Human vagina	3	56-78	No	N/A
Khan et al., 2016	Mouse ear and tail	2	48-65	No	N/A
Waise et al., 2019	Human tissue	3	56-75	No	N/A
Nadalutti et al., 2020	Human foreskin	1	65	No	N/A
Current	Human vagina	10	35-79	Yes	Spindle-shaped

Table 1: Comparison of Protocols for Successful Isolation of Human Vaginal Fibroblasts. Comparison of different existed protocols with ours and evidenced by morphological results.

Table 1 shows the results of using other protocols to attempt vaginal fibroblast isolation in this study. We developed a standard protocol for the isolation of primary fibroblasts from vaginal mucosa and older donors⁵.

We tested several established protocols, including those listed in Table 1, and observed no success in cell isolation using these protocols in our study. We propose the following reasons for their limitations.

The protocol published by Ruiz et al.³ involves scraping off fascia and cutting the tissue into small pieces. In our experience, it is hard to distinguish fascia, and attempts at scraping may result in a significant reduction in cell yield. While this method is straightforward, it lacks critical details, which limits its generalizability. Additionally, the mechanical digestion in this protocol appears insufficient for postmenopausal tissue, which tends to be denser.

Protocols published by Khan et al.⁹ and Waise et al.¹³ employ scissors for tissue mincing, which do not introduce significant multidirectional shearing forces compared to the scalpel technique. In our experience, the scissors method did not work effectively either.

Lastly, the Nadalutti et al.¹⁴ protocol, which used the explant method, did not successfully yield fibroblasts in our hands. This method may be less effective due to the nature of postmenopausal tissue, which may require more aggressive mechanical and enzymatic processing to dissociate fibroblasts successfully.

Figure 2: Phase contrast image of suspended cells on day 0. Please click here to view a larger version of this figure.

Figure 2 shows suspended cells on day 0 using our protocol.

Figure 3: Phase contrast image of vaginal primary fibroblasts on day 14 of culture at 100x magnification from a pooled cell suspension of three 1 cm² tissue biopsies. Please click here to view a larger version of this figure.

Figure 3 shows primary fibroblasts at 100x magnification from a pooled cell suspension of 3 tissue biopsies of 1 cm² in dimensions.

Fibroblast identity was investigated by immunofluorescent techniques as described earlier with minor modifications. To verify the cellular origin of primary vaginal cells, we used specific biomarkers of fibroblastic origin via immunofluorescent staining. Cells were identified as fibroblasts based on positive staining of vimentin (Figure 4), F-actin, and α-SMA from premenopausal and postmenopausal tissue samples (Figure 5). Fibroblasts were cultured to expansion with high viability (>90%) for use in experiments.

Figure 4: Positive staining of vimentin of vaginal fibroblasts. The isolated premenopausal and postmenopausal tissue primary fibroblasts were subjected to IF analysis and the images were taken at 200x magnification. Please click here to view a larger version of this figure.

Figure 5: Positive staining of F-actin and α-SMA of vaginal fibroblasts. The results of protein expression compared with IgG control. The images were collected at 200x magnification. Please click here to view a larger version of this figure.

Discussion

Many previous studies have reported how to isolate primary fibroblasts from the human vagina^3,7. Several studies used human vaginal tissue from premenopausal individuals with pelvic organ prolapse. Although a few papers reported the use of tissue from premenopausal and postmenopausal individuals^7,11, they did not describe in enough detail the protocol used to successfully isolate fibroblasts from older or postmenopausal donors. The vaginal wall is thicker in postmenopausal women with genital prolapse than in premenopausal women, which may account for the increased difficulty with isolation in this population¹³. Successful isolation from postmenopausal donors is essential for disease modeling and investigation, as pelvic floor disorders are most prevalent in the postmenopausal or older age groups.

We established a protocol to successfully and consistently harvest and culture human vaginal fibroblasts from donors aged 35-78 years old. The cellular origin of primary vaginal cells was confirmed by biomarkers of fibroblastic origin via immunofluorescent staining.

The human vaginal fibroblast isolation procedure presented here enables isolation and culture of primary fibroblast cells. In our experience, the use of previously published protocols for animal⁹and human^3,14,16 tissue for dissociation of primary cells failed to extract any fibroblasts from human vagina samples in this study¹⁴.

We hypothesized two probable reasons for challenges of cell dissociation from human vaginal tissue: (1) dermal thickness of mucosa is greater in older donors and when compared to that of non-mucosal tissue from older donors^13,17 making cell dissociation more difficult and (2) the density of fibroblasts may be markedly reduced in older individuals¹⁷.

Given these challenges, there are a few critical steps in this protocol that should not be modified. The first critical step is the implementation of a very rigorous mechanical digestion. Here, we use the two-scalpel technique. In our experience, substitution with scissors to mince the tissue does not yield small enough fragments to dissociate fibroblasts from human vaginal tissue.

The other critical step is pooling the cell suspension and pooling of 3-4 full thickness biopsies (1 cm²) from a single donor. This is necessary to obtain sufficient cells for ongoing cultivation. In all cases, we harvested significantly more tissue than 1 cm² as excess vaginal epithelium is trimmed as a necessary part of pelvic organ prolapse surgery. In this study, we only pooled cell suspensions that originated from the same donor as we sought to investigate and differentiate cellular characteristics and proliferation between various donors.

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. The need for pooling of cell suspensions of multiple tissue biopsy samples may also be a limitation.

In conclusion, this protocol for acquiring human vaginal fibroblasts will be a useful tool for future investigations in pelvic floor disorders, especially in postmenopausal individuals as well as an important addition to women's health research.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amphotericin B	Bio Techne	B23192
Cell strainer (100 μm)	ThermoFisher Scientific	22363549
Conical Centrifuge Tubes (15 mL)	Fisher Scientific	14-959-53A
DMEM/F12	ThermoFisher Scientific	11320033
Feather Disposable Scalpel No. 15	Socorex	FB.15
Fetal Bovine Serum	Sigma Aldrich	NC1983075
High Clarity Conical Centrifuge Tubes (50 mL)	Fisher Scientific	14-432-22
HulaMixer Sample Mixer	ThermoFisher Scientific	15920D
Human Fibronectin DuoSet ELISA	R&D Systems	DY1918-05
Human Pro-Collagen I alpha 1 DuoSet ELISA	R&D Systems	DY6220-05
Liberase Research Grade	Sigma Aldrich	05 401 119 001
Penicillin/Streptomycin (5000U/ml)	ThermoFisher Scientific	15070063
Petri dishes, polystyrene (100 mm x 20 mm)	Millipore Sigma	P5606
Serologic pipettes (10 mL)	ThermoFischer Scientific	170356N
Sterile syringes (5 mL)	Fischer Scientific	14-955-458