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 findings: Pelvic Organ Prolapse Quantification (POP-Q) measurements were obtained in the clinic, showing evidence of pelvic organ prolapse.</li>
	<li>Use the following inclusion criteria: over 18 years of age; history of symptomatic pelvic organ prolapse; electing for surgical correction of pelvic organ prolapse.</li>
	<li>Exclude the following: Pregnant women and patients refusing to donate tissue.</li>
	<li>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.</li>
	<li>Transport tissue to the lab in a sterile specimen collection cup with normal saline or serum-free DMEM/F12 (Gibco) media. Keep on ice.</li>
</ol>
2. Tissue processing and digestion
<ol>
	<li>Tissue preparation
	<ol>
		<li>Measure and cut the tissue to encompass the full thickness of the vaginal epithelium, creating segments of approximately 1 cm2.</li>
		<li>Ensure precise measurement of the biopsy size 1 cm2. 
		NOTE: Overestimating the tissue biopsy size may impair tissue digestion.</li>
		<li>Prepare 2-3 additional vaginal biopsies measuring 1 cm2 each from a single donor and set the biopsies aside.</li>
	</ol>
	</li>
	<li>Mechanical digestion
	<ol>
		<li>Place the vaginal biopsy in a 10 cm cell culture Petri dish. Pipette 500-1,000 &#181;L of serum-free media supplemented with 100 U/mL penicillin/streptomycin, 2.5 &#181;g/mL amphotericin Bonto the vaginal tissue to keep the tissue from drying out.</li>
		<li>Mince the vaginal tissue into small fragments using two sterile scalpels (No. 11 or No. 15) in both hands.</li>
		<li>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.</li>
		<li>Repeat this mincing technique using the two-scalpel technique until the sample is of uniform consistency with 1-2 mm pieces. 
		&#8203;NOTE: Mechanical digestion using this two-scalpel technique of a 1 cm2 biopsy should take approximately 15-20 min to complete.</li>
		<li>Add 2-3 mL of serum-free DMEM/F12 media supplemented with 100 U/mL penicillin/streptomycin, 2.5 &#181;g/mL amphotericin B to the plate, suspending the minced tissue. Pipette the solution containing tissue fragments up and down to break up any clumps.</li>
	</ol>
	</li>
	<li>Enzymatic digestion
	<ol>
		<li>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.</li>
		<li>Dissolve 5 mg of Liberase in 1 mL of sterile water (5 mg/mL). Store in 230 &#181;L aliquots at &#8722;20 &#176;C for up to a month or &#8722;80 &#176;C for 6 months.</li>
		<li>Add 230 &#181;L 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.</li>
		<li>Repeat steps 2.1-2.3 for 2-3 additional vaginal biopsies of the same donor. 
		&#8203;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.</li>
		<li>Incubate the tubes for 3 h at 37 &#176;C with constant, vigorous agitation using a sample mixer.</li>
		<li>Place a sample mixer in a CO2 incubator. Maintain constant agitation. (Sample mixer settings: 25 rpm of rotational motion for 5 s, 30&#176; of reciprocal (tilting rotation) for 5 s, and 2&#176; of vibration (vortexing) motion for 3 s).</li>
		<li>During the incubation, vortex the tubes at 30 min intervals.</li>
		<li>Centrifuge the samples at 3,000 x g for 5 min. Remove and discard the supernatant.</li>
		<li>Resuspend the pellet in 1-2 mLof DMEM/F12 media with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin, 2.5 &#181;g/mL amphotericin B to dilute the Liberase enzyme. Vigorously pipette until the pellet is fully resuspended.</li>
	</ol>
	</li>
</ol>
3. Cell culture
<ol>
	<li>Removal of non-digested tissue fragments
	<ol>
		<li>Place a 100 &#181;m cell strainer over a sterile 50 mL conical container.</li>
		<li>Pool the cell suspension from the multiple tissue biopsies of the same donor together.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Cell culture pooling
	<ol>
		<li>Plate pooled cell suspension from multiple vaginal biopsies (from the same donor) on a Petri dish (60 mm x 15 mm).</li>
		<li>Incubate the Petri dish in a CO2 incubator at 37 &#176;C overnight.</li>
		<li>Observe and confirm the presence of non-adherent cells with a phase-contrast microscope.</li>
		<li>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.</li>
		<li>Wait for 18-24 h prior to changing cell culture media to ensure adequate cell adherence.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol><li>Mirin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gender+disparity+in+the+funding+of+diseases+by+the+U.S%5BTitle%5D)+AND+%22J+Womens+Health%22%5BJournal%5D)">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/pubmed?term=((The+prevalence+of+pelvic+floor+disorders+and+their+relationship+to+gender%2C+age%2C+parity%2C+and+mode+of+delivery%5BTitle%5D)+AND+%22BJOG%22%5BJournal%5D)">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., Brölmann, H. A. M., Smit, T. H., Helder, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblasts+from+women+with+pelvic+organ+prolapse+show+differential+mechanoresponses+depending+on+surface+substrates%5BTitle%5D)+AND+%22Int+Urogynecol+J+Pelvic+Floor+Dysfunct%22%5BJournal%5D)">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/pubmed?term=((Cell+lines%5BTitle%5D)+AND+%22Spermatogenesis%22%5BJournal%5D)">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/pubmed?term=((Maintenance+of+chronological+aging+features+in+culture+of+normal+human+dermal+fibroblasts+from+old+donors%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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/pubmed?term=((Generating+primary+murine+vaginal+fibroblast+cell+lines%5BTitle%5D)+AND+%22MethodsX%22%5BJournal%5D)">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/pubmed?term=((comparative+characterization+of+vaginal+cells+derived+from+premenopausal+women+with+and+without+severe+pelvic+organ+prolapse%5BTitle%5D)+AND+%22Reprod+Sci%22%5BJournal%5D)">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/pubmed?term=((Vaginal+fibroblastic+cells+from+women+with+pelvic+organ+prolapse+produce+matrices+with+increased+stiffness+and+collagen+content%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((Generating+primary+fibroblast+cultures+from+mouse+ear+and+tail+tissues%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Reprod+Sci%22%5BJournal%5D)">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/pubmed?term=((Attachment+of+primary+vaginal+fibroblasts+to+absorbable+and+nonabsorbable+implant+materials+coated+with+platelet-rich+plasma%5BTitle%5D)+AND+%22Female+Pelvic+Med+Reconstr+Surg%22%5BJournal%5D)">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/pubmed?term=((Prevalence%2C+risk+factors%2C+and+predictors+of+pelvic+organ+prolapse%5BTitle%5D)+AND+%22Menopause%22%5BJournal%5D)">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/pubmed?term=((An+optimized+method+to+isolate+human+fibroblasts+from+tissue+for+ex+vivo+analysis%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">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/pubmed?term=((Using+human+primary+foreskin+fibroblasts+to+study+cellular+damage+and+mitochondrial+dysfunction%5BTitle%5D)+AND+%22Curr+Protoc+Toxicol%22%5BJournal%5D)">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/pubmed?term=((Menopause+Leading+to+Increased+Vaginal+Wall+Thickness+in+Women+with+Genital+Prolapse%3A+Impact+on+Sexual+Response%5BTitle%5D)+AND+%22J+Sex+Med%22%5BJournal%5D)">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/pubmed?term=((Measurement+of+oral+epithelial+thickness+by+optical+coherence+tomography%5BTitle%5D)+AND+%22Diagnostics%22%5BJournal%5D)">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/pubmed?term=((Age-related+changes+in+the+fibroblastic+differon+of+the+dermis%3A+role+in+skin+aging%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">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>

We have no conflicts of interest to disclose.

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 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.&#160;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 human3,14,16 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 donors13,17 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&#39;s health research.

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><tbody><tr><td>Amphotericin B</td><td>Bio Techne</td><td>B23192</td><td></td></tr><tr><td>Cell strainer (100 &amp;mu;m)</td><td>ThermoFisher Scientific</td><td>22363549</td><td></td></tr><tr><td>Conical Centrifuge Tubes (15 mL)</td><td>Fisher Scientific&amp;nbsp;</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&amp;nbsp;</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&amp;amp;D Systems</td><td>DY1918-05</td><td></td></tr><tr><td>Human Pro-Collagen I alpha 1 DuoSet ELISA</td><td>R&amp;amp;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) 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65864/65864fig01.jpg" /> 
Figure 1: Diagrammatic representation of the protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65864/65864fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 1&#160;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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protocol (Author, year)</td>
			<td>Donor tissue of original protocol</td>
			<td>Number of Donors</td>
			<td>Donor age in years</td>
			<td>Fibroblasts obtained</td>
			<td>Morphology</td>
		</tr>
		<tr>
			<td>Ruiz-Zapata et al., 2013</td>
			<td>Human vagina</td>
			<td>3</td>
			<td>56-78</td>
			<td>No</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Khan et al., 2016</td>
			<td>Mouse ear and tail</td>
			<td>2</td>
			<td>48-65</td>
			<td>No</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Waise et al., 2019</td>
			<td>Human tissue</td>
			<td>3</td>
			<td>56-75</td>
			<td>No</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Nadalutti et al., 2020</td>
			<td>Human foreskin</td>
			<td>1</td>
			<td>65</td>
			<td>No</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Current</td>
			<td>Human vagina</td>
			<td>10</td>
			<td>35-79</td>
			<td>Yes</td>
			<td>Spindle-shaped</td>
		</tr>
	</tbody>
</table>
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.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65864/65864fig02.jpg" /> 
Figure 2: Phase contrast image of suspended cells on day 0. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65864/65864fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2&#160;shows suspended cells on day 0 using our protocol.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65864/65864fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65864/65864fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3 shows primary fibroblasts at 100x magnification from a pooled cell suspension of 3 tissue biopsies of 1 cm&#178; 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 &#945;-SMA from premenopausal and postmenopausal tissue samples (Figure 5). Fibroblasts were cultured to expansion with high viability (&#62;90%) for use in experiments.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65864/65864fig04.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65864/65864fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65864/65864fig05.jpg" /> 
Figure 5: Positive staining of F-actin and &#945;-SMA of vaginal fibroblasts. The results of protein expression compared with IgG control. The images were collected at 200x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65864/65864fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

348 Views.  Massachusetts General Hospital. Onderzoek naar aandoeningen die vooral mensen van het vrouwelijk geslacht treffen, is onderbelicht en ondergefinancierd. Verzakking van het bekkenorgaan is de aandoening die sterk wordt geassocieerd met het vrouwelijk geslacht. Het treedt op wanneer spieren en ligamenten verzwakken en ervoor zorgen dat bekkenorganen lager in het bekken vallen, waardoor een uitstulping in de vagina ontstaat.Er is weinig bekend over cellulaire interacties in de context v...

Video: Weefselverwerking en isolatie van primaire fibroblasten uit de menselijke vagina

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Developmental Biology

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Medicine

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetics

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioengineering

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Neuroscience

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina

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