We would like to express our&#160;gratitude to all members of the Vincent Center&#160;of Reproductive Biology at Massachusetts&#160;General Hospital, as well as the VIncent Memorial Hospital Foundation, for their&#160;generous support. Special thanks to Dr. Bo&#160;Rueda and his lab for their valuable suggestions&#160;and for lending us lab equipment.

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
<ol>
	<li>Diagnose pelvic organ prolapse by taking a history and using pelvic exam find...

<ol>
	<li>Mirin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+disparity+in+the+funding+of+diseases+by+the+U.S.">Gender disparity in the funding of diseases by the U.S.</a> J Womens Health. 30 (7), 956-963 (2021).</li><li>MacLennan, A. H., Taylor, A. W., Wilson, D. H., Wilson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+pelvic+floor+disorders+and+their+relationship+to+gender,+age,+parity,+and+mode+of+delivery.">The prevalence of pelvic floor disorders and their relationship to gender, age, parity, and mode of delivery.</a> BJOG. 107 (12), 1460-1470 (2000).</li><li>Ruiz-Zapata, A. M., Kerkhof, M. H., Zandieh-Doulabi, B., Br&#246;lmann, H. A. M., Smit, T. H., Helder, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+from+women+with+pelvic+organ+prolapse+show+differential+mechanoresponses+depending+on+surface+substrates.">Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates.</a> Int Urogynecol J Pelvic Floor Dysfunct. 24 (9), 1567-1575 (2013).</li><li>Kaur, G., Dufour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines.">Cell lines.</a> Spermatogenesis. 2 (1), 1-5 (2012).</li><li>Rorteau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+chronological+aging+features+in+culture+of+normal+human+dermal+fibroblasts+from+old+donors.">Maintenance of chronological aging features in culture of normal human dermal fibroblasts from old donors.</a> Cells. 11 (5), 858 (2022).</li><li>Blum, M., Koehler, J., Yangdon, T., Chatterjea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+primary+murine+vaginal+fibroblast+cell+lines.">Generating primary murine vaginal fibroblast cell lines.</a> MethodsX. 7, 101100 (2020).</li><li>Kufaishi, H., Alarab, M., Drutz, H., Lye, S., Shynlova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=comparative+characterization+of+vaginal+cells+derived+from+premenopausal+women+with+and+without+severe+pelvic+organ+prolapse.">comparative characterization of vaginal cells derived from premenopausal women with and without severe pelvic organ prolapse.</a> Reprod Sci. 23 (7), 931-943 (2016).</li><li>Ruiz-Zapata, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaginal+fibroblastic+cells+from+women+with+pelvic+organ+prolapse+produce+matrices+with+increased+stiffness+and+collagen+content.">Vaginal fibroblastic cells from women with pelvic organ prolapse produce matrices with increased stiffness and collagen content.</a> Sci Rep. 6 (1), 22971 (2016).</li><li>Khan, M., Gasser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+primary+fibroblast+cultures+from+mouse+ear+and+tail+tissues.">Generating primary fibroblast cultures from mouse ear and tail tissues.</a> J Vis Exp. (107), e53565 (2016).</li><li>Kufaishi, H., Alarab, M., Drutz, H., Lye, S., Shynlova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+mechanical+loading+influences+the+expression+of+extracellular+matrix+and+cell+adhesion+proteins+in+vaginal+cells+derived+from+premenopausal+women+with+severe+pelvic+organ+prolapse.">Static mechanical loading influences the expression of extracellular matrix and cell adhesion proteins in vaginal cells derived from premenopausal women with severe pelvic organ prolapse.</a> Reprod Sci. 23 (8), 978-992 (2016).</li><li>Medel, S., Alarab, M., Kufaishi, H., Drutz, H., Shynlova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+primary+vaginal+fibroblasts+to+absorbable+and+nonabsorbable+implant+materials+coated+with+platelet-rich+plasma.">Attachment of primary vaginal fibroblasts to absorbable and nonabsorbable implant materials coated with platelet-rich plasma.</a> Female Pelvic Med Reconstr Surg. 21 (4), 190-197 (2015).</li><li>Awwad, J., Sayegh, R., Yeretzian, J., Deeb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+risk+factors,+and+predictors+of+pelvic+organ+prolapse.">Prevalence, risk factors, and predictors of pelvic organ prolapse.</a> Menopause. 19 (11), 1235-1241 (2012).</li><li>Waise, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+to+isolate+human+fibroblasts+from+tissue+for+ex+vivo+analysis.">An optimized method to isolate human fibroblasts from tissue for ex vivo analysis.</a> Bio Protoc. 9 (23), e3440 (2019).</li><li>Nadalutti, C. A., Wilson, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+human+primary+foreskin+fibroblasts+to+study+cellular+damage+and+mitochondrial+dysfunction.">Using human primary foreskin fibroblasts to study cellular damage and mitochondrial dysfunction.</a> Curr Protoc Toxicol. 86 (1), (2020).</li><li>Da Silva Lara, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menopause+Leading+to+Increased+Vaginal+Wall+Thickness+in+Women+with+Genital+Prolapse:+Impact+on+Sexual+Response.">Menopause Leading to Increased Vaginal Wall Thickness in Women with Genital Prolapse: Impact on Sexual Response.</a> J Sex Med. 6 (11), 3097-3110 (2009).</li><li>Stasio, D. D., Lauritano, D., Iquebal, H., Romano, A., Gentile, E., Lucchese, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+oral+epithelial+thickness+by+optical+coherence+tomography.">Measurement of oral epithelial thickness by optical coherence tomography.</a> Diagnostics. 9 (3), 90 (2019).</li><li>Zorina, A., Zorin, V., Kudlay, D., Kopnin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+the+fibroblastic+differon+of+the+dermis:+role+in+skin+aging.">Age-related changes in the fibroblastic differon of the dermis: role in skin aging.</a> Int J Mol Sci. 23 (11), 6135 (2022).</li></ol>

Many previous studies have reported how to isolate primary fibroblasts from the human vagina3,7. 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 individuals7,11, they did not describe in enough detail the protocol used to successfully isolate fibroblasts from older or...

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 models7,8. 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 prolapse7,10. 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 &#945;-smooth muscle actin (&#945;-SMA).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Bio Techne</td>
			<td>B23192</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (100 &#956;m)</td>
			<td>ThermoFisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes (15 mL)</td>
			<td>Fisher Scientific&#160;</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Disposable Scalpel No. 15</td>
			<td>Socorex</td>
			<td>FB.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>NC1983075</td>
			<td></td>
		</tr>
		<tr>
			<td>High Clarity Conical Centrifuge Tubes (50 mL)</td>
			<td>Fisher Scientific&#160;</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>HulaMixer Sample Mixer</td>
			<td>ThermoFisher Scientific</td>
			<td>15920D</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Fibronectin DuoSet ELISA</td>
			<td>R&#38;D Systems</td>
			<td>DY1918-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Pro-Collagen I alpha 1 DuoSet ELISA</td>
			<td>R&#38;D Systems</td>
			<td>DY6220-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase Research Grade</td>
			<td>Sigma Aldrich</td>
			<td>05 401 119 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (5000U/ml)</td>
			<td>ThermoFisher Scientific</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene (100 mm x 20 mm)</td>
			<td>Millipore Sigma</td>
			<td>P5606</td>
			<td></td>
		</tr>
		<tr>
			<td>Serologic pipettes (10 mL)</td>
			<td>ThermoFischer Scientific</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringes (5 mL)</td>
			<td>Fischer Scientific</td>
			<td>14-955-458</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue processing and isolation of primary fibroblasts from the human vagina

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.&#160;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.

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) ...

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.

Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina

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 ...

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.

tissue-processing-isolation-primary-fibroblasts-from-human

Research

JoVE Journal

Biology

128 visualizações.  Massachusetts General Hospital. A pesquisa sobre distúrbios que afetam principalmente pessoas do sexo feminino é pouco estudada e subfinanciada. O prolapso de órgãos pélvicos é o distúrbio fortemente associado ao sexo feminino. Ocorre quando músculos e ligamentos enfraquecem e fazem com que os órgãos pélvicos caiam mais baixo na pelve, criando uma protuberância na vagina.Pouco se sabe sobre as interações celulares no contexto dessa fisiopatologia, nem como os fatores no...