Name: ultrasonic-augmented primary adult fibroblast isolation
Uploaded: 2019-07-29T14:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Ultrasonic-augmented Primary Adult Fibroblast Isolation at JoVE.com

We thank Ms. Romy Kempe and Mrs. Annett Opitz for expert technical support. We also thank Mr. Bjoern Binnewerg for IT support. This work was supported by grants from a) the F&#246;rderkreis Dresdner Herz-Kreislauf-Tage e.V., b) &#34;Habilitationsf&#246;rderprogramm f&#252;r Frauen&#34;, Faculty of Medicine Carl Gustav Carus Dresden and c) Else Kr&#246;ner-Forschungskolleg (EKFK) Faculty of Medicine Carl Gustav Carus Dresden. We are grateful for the funding and the support.

The following protocol follows the institutional animal care guidelines of Technische Universit&#228;t Dresden, Germany (File number: T 2014/4) as well as internationally accepted animal care guidelines (FELASA)17. Figure 1 visualizes the cell isolation process.
1. Preparing the setup, material and media
<ol>
	<li>Prepare cell culture medium, PBS solution, collagenase blend stock solution (reconstitute 50 mg of lyophilized collagenase blend in 12 mL of sterile ultrapure water), and 0.25% trypsin solution.</li>
	<li>Warm up the medium, the PBS and the trypsin solution to 37 &#176;C.</li>
	<li>Preheat the ultrasonic water bath to 37 &#176;C.</li>
	<li>Disinfect forceps, a stainless-steel spatula, scalpels (2x scalpels per organ) and 2 glass beakers with 70% ethanol and place these materials under the cell culture hood.</li>
	<li>Fill one beaker with 70% ethanol and the other with sterile water or PBS solution. These beakers are required to disinfect and wash the instrument after each organ procession.</li>
	<li>Place sterile 15 mL plastic tubes containing cold PBS on wet ice. The number of tubes depends on the number of organs you want to isolate fibroblasts from.</li>
</ol>
2. Mouse dissection and organ removal
<ol>
	<li>Wear two pairs of gloves one above the other, so the first pair can be removed as soon as the animal has been dissected. 
	NOTE: This procedure prevents bacteria from the animal&#39;s fur and skin from spreading over the organs.</li>
	<li>Euthanize the mouse (e.g., cervical dislocation) and pin the carcass with needles to every limb to a polystyrene pad.</li>
	<li>Disinfect the mouse carcass using 70% ethanol spray. Make sure the fur is soaked in ethanol so the hair will not swirl up.</li>
	<li>Cut the fur right above the urogenital tract using surgical forceps and atraumatic scissors. Cut the skin alongside the middle line from the point of the initial incision to the neck (3 -&#160;4 cm) and add relief cuts at the limbs. 
	CAUTION: Do not perforate the muscular layer at this step to avoid bacterial contamination!</li>
	<li>Pin the skin to the polystyrene foam pad to have optimal access to the musculature covering the abdominal cavity.</li>
	<li>Disinfect the abdominal musculature twice using 70% ethanol. Let the ethanol dry before continuing to the next step.</li>
	<li>Remove the first pair of gloves. Use a new, sterile set of forceps and scissors.</li>
	<li>Open the abdominal cavity and the thorax by incising the muscular layer with surgical scissors to gently remove the organs of choice. Therefore, hold the organ gently with surgical forceps (do not pierce the organ, use minimal pressure) and cut the supplying blood vessels near the entry point at the organ with scissors.</li>
	<li>Put the organs into the sterile tubes containing cold PBS. Close the tubes tightly. Place the tubes on wet ice until continuing with step 3.1.</li>
</ol>
3. Tissue mincing, digestion and cell extraction
<ol>
	<li>Transfer the tubes under the sterile cell culture hood. 
	CAUTION: Wear a fresh pair of gloves and disinfect the tubes with 70% ethanol before transferring them under the hood!</li>
	<li>Take the organ out of the 15 mL tube using sterile forceps. Place the organ onto one half of a sterile 6 cm Petri dish and wash the organ briefly with PBS to remove excess blood. Transfer the organ to the second half of the Petri dish, remove excess PBS.</li>
	<li>Mince the tissue using two sterile scalpels. The remaining tissue fragments should not be larger than 1 -&#160;2 mm.</li>
	<li>Transfer the minced tissue into a new sterile 15 mL tube using the sterile spatula and add 2 mL of 0.25% trypsin solution. Place the tube into a cell culture incubator at 37 &#176;C for 5 min.</li>
	<li>Vortex the tube gently (circa 1400/min) for 10 s.</li>
	<li>Stop the trypsin reaction under the cell culture hood by adding 4 mL FCS-containing cell culture medium (Dulbecco&#39;s Modified Eagle Medium (DMEM), e.g.).</li>
	<li>Add 250 &#181;L of collagenase blend solution to each tube containing heart or lung tissue and 100 &#181;L for kidney or liver, respectively.</li>
	<li>Place the tubes into an ultrasonic water bath (37 &#176;C) and activate the ultrasonic sonicator for 10&#160;min. 
	NOTE: The ultrasonic water bath used in this protocol has an operating frequency of 35 kHz and a maximal power of 320 W.</li>
	<li>Vortex the tubes gently (circa 1400/min) for 10 s.</li>
	<li>Place the tubes again into the ultrasonic water bath for 10 min.</li>
	<li>Vortex gently (circa 1400/min) for 10 s.</li>
	<li>Disinfect the tubes with 70% ethanol and transfer them under the sterile cell culture hood.</li>
	<li>Filter the solution with a 40 &#181;m mesh into a new sterile 15 mL tube.</li>
	<li>Centrifuge the tube at 500 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of fresh medium.</li>
	<li>Transfer the cells into a suitable cell culture vessel (e.g., 6-well plate) and place the vessel into the cell culture incubator overnight at 37 &#176;C and 5% CO2.</li>
	<li>The next day, remove the medium, wash 3 times with PBS, then add fresh medium (the added volume depends on the cell culture vessel of choice, 2 mL per well of a 6-well plate etc.).</li>
	<li>Change the medium every other day. 
	NOTE: Fibroblasts can be split after reaching optical confluence of 90% (usually after 5-7 days).</li>
</ol>

<ol>
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Cardiology. 14 (8), 484-491 (2017).</li><li>Weissman-Shomer, P., Fry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+fibroblasts+senscence+in+vitro:+pattern+of+cell+division+and+life+span+as+a+function+of+cell+density.">Chick embryo fibroblasts senscence in vitro: pattern of cell division and life span as a function of cell density.</a> Mechanisms of Ageing and Development. 4 (2), 159-166 (1975).</li><li>Angello, J. 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W., Aslam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Isolation+of+High+Quality+Cardiomyocytes,+Endothelial+Cells,+and+Fibroblasts+from+an+Adult+Rat+Heart.">Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart.</a> Journal of Visualized Experiments. (123), e55601 (2017).</li><li>Weldrick, J. J., Abdul-Ghani, M., Megeney, L. A., Burgon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+the+isolation+of+postnatal+murine+cardiac+myocyte+and+fibroblast+cells.">A rapid and efficient method for the isolation of postnatal murine cardiac myocyte and fibroblast cells.</a> Canadian Journal of Physiology and Pharmacology. 96 (5), 535-539 (2018).</li><li>Wang, H., Van Blitterswijk, C. A., Bertrand-De Haas, M., Schuurman, A. H., Lamme, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+enzymatic+isolation+of+fibroblasts+for+the+creation+of+autologous+skin+substitutes.">Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.</a> In Vitro Cellular &amp; Developmental Biology. Animal. 40 (8-9), 268-277 (2004).</li><li>El-Armouche, 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=Phosphatase+inhibitor-1-deficient+mice+are+protected+from+catecholamine-induced+arrhythmias+and+myocardial+hypertrophy.">Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy.</a> Cardiovascular Research. 80 (3), 396-406 (2008).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+Guidelines+and+Recommendations.">FELASA Guidelines and Recommendations.</a> Journal of the American Association for Laboratory Animal Science. 51 (3), 311-321 (2012).</li><li>Seluanov, A., Vaidya, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> Journal of Visualized Experiments. (44), 2033 (2010).</li><li>Masur, S. K., Dewal, H. S., Dinh, T. T., Erenburg, I., Petridou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblasts+differentiate+from+fibroblasts+when+plated+at+low+density.">Myofibroblasts differentiate from fibroblasts when plated at low density.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (9), 4219-4223 (1996).</li><li>Rohr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblasts+in+cell+culture+systems:+myofibroblasts+all+along.">Cardiac fibroblasts in cell culture systems: myofibroblasts all along.</a> Journal of Cardiovascular Pharmacology. 57 (4), 389-399 (2011).</li><li>Cell lines: Valuable tools or useless artifacts. PubMed - NCBI Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22553484">https://www.ncbi.nlm.nih.gov/pubmed/22553484</a> (2019)</li><li>Copp&#233;, J. P., 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS biology. 6 (12), 2853-2868 (2008).</li><li>Childs, B. G., Durik, M., Baker, D. J., van Deursen, J. 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There are no conflicts of interest to declare.

Compared to immortalized fibroblast cell lines, primary fibroblasts offer several advantages. They can be isolated cost effectively in high quality and quantity. Furthermore, primary cultures offer the possibility to study cells from multiple individuals, which increases the reliability of the obtained results and decreases the likelihood of merely studying cell culture artifacts. Continuous generation of new primary cultures prevents genetic alterations which commonly occur after repeated passaging21</...

Fibroblasts are flat, spindle-shaped cells with multiple stellate processes and an extensive rough endoplasmic reticulum1,2. An average fibroblast measures 30 -&#160;100 &#181;m and has a life span of 57 &#177; 3 days1,3. The average cell cycle duration of human fibroblasts ranges from 16 -&#160;48 h depending on the culture conditions4. There is evidence that the replicative capacity and functional quality of cultured primary fibroblasts negatively correlates with the donor age, suggesting that younger donors (animals or patients) should be preferred if possible5,6.
Fibroblasts constitute a predominant cell type of most mammalian body tissues. Despite their ubiquitous presence, the molecular identification of fibroblasts is still a challenge7. Fibroblasts migrate to developing tissues and organs from different sources during embryonic development8. For this reason, there is a plethora of marker proteins that can be found in fibroblasts whereas unique marker proteins, which are present in every fibroblast population and exclusive for fibroblasts, are still missing. Thus, expression patterns of several recognized markers are usually used to identify fibroblasts. Among the most recognized markers are vimentin, human fibroblast surface protein (hFSP), discoidin domain receptor 2 (DDR2) and alpha smooth muscle actin (&#945;SMA).
Fibroblasts are the major extracellular matrix (ECM)-producing cell type. Thereby, fibroblasts maintain an orderly tissue architecture and provide mechanical support for neighboring cells1. The balance between ECM synthesis and degradation is a well-regulated process. Shifts towards synthesis mark the beginning of excessive ECM deposition which, if not terminated, leads to fibrosis. Fibrosis is mediated by myofibroblasts, which originate from activated fibroblasts undergoing molecular and phenotypical changes. One hallmark of myofibroblasts is enhanced secretion of ECM and cytokines and the expression of orderly arranged &#945;SMA microfilaments9.
Primary fibroblasts have been in the spotlight of recent research focusing on fibrosis, tissue inflammation and fibroblast-cancer-cell interactions10,11. However, to effectively study fibroblast properties in health and disease, it is necessary to isolate viable primary adult fibroblasts on a regular basis. There are several methods available to isolate fibroblasts12,13,14. The three major methods of fibroblast isolation are outgrowth from tissue chunks12, enzymatic tissue digestion15, and enzymatic perfusion of hollow organs9,13,16. The advantage of outgrowth is a gentle isolation process without enzymatic cell degradation. On the other hand, outgrowth cultures usually require prolonged culture periods until cells can be used for experiments. Common enzymatic digestion is fast but bears a risk of contamination with other cell types (e.g., endothelial cells) or bacteria in the agitation process, which is necessary to mechanically dissolve the tissue. Furthermore, these methods are often elaborate and require time and skill to learn.
Regarding the importance of primary fibroblasts in research, there is still a need to optimize existing cell isolation approaches in terms of quickness, simplicity and reliability. Here, a novel ultrasonic-based enzymatic fibroblast isolation method delivering high quality cells is provided.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Sigma-Aldrich, St. Louis, USA</td><td>T4049-100ML</td><td></td></tr><tr><td>Antibiotics</td><td>Gibco-Life Technologies, Carlsbad, USA</td><td>Gibco LS15140148&amp;nbsp;</td><td>Penicillin/ Streptomycin (10,000 U/mL)</td></tr><tr><td>Cell culture hood</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>51023608</td><td>HeraSafe KSP15</td></tr><tr><td>Cell culture incubator</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>50049176</td><td>BBD 6220</td></tr><tr><td>Cell culture plates</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>depends on vessel</td><td>6-, 12-, 24-wells Nunclon surface</td></tr><tr><td>Cell culture suction</td><td>VACUUBRAND GMBH + CO KG, Wertheim, Germany</td><td>20727400</td><td>BVC professional suction</td></tr><tr><td>Cell strainer (mesh)</td><td>Corning, Tewksbury, USA</td><td>431750</td><td>40 &amp;micro;m Nylon</td></tr><tr><td>Centrifuge</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>75007213</td><td>Megafuge 8R</td></tr><tr><td>Cordless pipetting controller</td><td>Hirschmann, Eberstadt, Germany</td><td>9907200</td><td>Pipetus</td></tr><tr><td>Disposable pipette tips</td><td>Sigma-Aldrich, St. Louis, USA</td><td>depends on volume</td><td>SafeSeal tips for pipettes (10 &amp;micro;L, 20 &amp;micro;L, 100 &amp;micro;L, 200 &amp;micro;L, 1000 &amp;micro;L)</td></tr><tr><td>Disposable plastic pipettes</td><td>Sigma-Aldrich, St. Louis, USA</td><td>depends on volume</td><td>5 mL, 10 mL, 25 mL, 50 mL</td></tr><tr><td>Disposable sterile scalpel</td><td>Myco Medical, Cary, USA</td><td>n.a.</td><td>Techno cut</td></tr><tr><td>Dulbeccos Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>41965-062</td><td>High glucose</td></tr><tr><td>Eppendorf tubes</td><td>Eppendorf, Hamburg, Germany&amp;nbsp;</td><td>depends on volume</td><td>50 &amp;micro;L, 500 &amp;micro;L, 1.500&amp;micro;L, 2.000 &amp;micro;L</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>F2442-50ML</td><td></td></tr><tr><td>Collagenase blend</td><td>Sigma-Aldrich, St. Louis, USA</td><td>5401020001</td><td>Liberase TL Research Grade</td></tr><tr><td>Petri dish 6 cm</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P5481-500EA</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>D8537-500ML</td><td>500 mL</td></tr><tr><td>Senescence detection kit</td><td>Abcam, Cambridge, UK</td><td>ab65351</td><td></td></tr><tr><td>Shaker/ Vortex</td><td>IKA, Staufen im Breisgau, Germany</td><td>n.a.</td><td>MS2 Minishaker (subsequent model: Ident-Nr.: 0020016017)</td></tr><tr><td>Sterile plastic tubes</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>Falcon 352095</td><td>BD Falcon tubes (15 mL, 50 mL)</td></tr><tr><td>Ultrasonic water bath</td><td>BANDELIN electronic GmbH &amp;amp; Co. KG, Berlin, Germany</td><td>312</td><td>Sonorex RK100H</td></tr><tr><td>Surgical scissors (atraumatic)</td><td>Aesculap AG, Tuttlingen, Germany</td><td>NR 82</td><td></td></tr><tr><td>Surgical scissors&amp;nbsp;</td><td>Aesculap AG, Tuttlingen, Germany</td><td>eq 1060.09</td><td></td></tr><tr><td>Surgical forceps</td><td>Aesculap AG, Tuttlingen, Germany</td><td>BD577</td><td></td></tr></tbody></table>

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.

The ability of this protocol to isolate adult fibroblasts from solid murine tissue was demonstrated. Viable fibroblasts were obtained that could be used for subsequent experiments such as immunofluorescence staining or proliferation experiments (Figure 2D-F, Figure 5A).
Adult fibroblasts are flat spindle-shaped cells with multiple cellular processes that typically grow in monolayers1...

Watch this Scientific Journal Video about Ultrasonic-augmented Primary Adult Fibroblast Isolation at JoVE.com

Ultrasonic-augmented Primary Adult Fibroblast Isolation

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 ...

ultrasonic-augmented-primary-adult-fibroblast-isolation

Research

JoVE Journal

Medicine

7.4K Views.  Technische Universität Dresden. 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).

Video: Ultrasonic-augmented Primary Adult Fibroblast Isolation

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 ...

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

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Cancer Research

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Biology

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

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

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, 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 ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...