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>

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	<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>

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...

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

231 Views.  Massachusetts General Hospital. 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.

Video: Tissue Processing and Isolation of Primary Fibroblasts from the Human Vagina

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...