Name: Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts (Video) | JoVE Journal
Uploaded: 2025-08-01T14:25:00.000+00:00
Duration: 9 min 43 s
Description: Learn to isolate, culture, and characterize prostate cancer-associated fibroblasts from radical prostatectomy specimens. This method enables reliable generation of CAFs for studying tumor-stromal interactions and therapeutic targets.

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This work was supported by the Italian Cancer Research Association (AIRC),&#160;IG16930&#160;and IG24851 to V.P; the Italian Ministry of University and Research (MIUR PRIN 2017 and 2022 to V.P.); the Truus and Gerrit van Riemsdijk Foundation, Liechtenstein, donation to V.P.; Piedmont Region (Deflect). A.S. was supported by an Italian Cancer Research Foundation (FIRC) post-doctoral fellowship, and L.A. was supported by Fondazione Umberto Veronesi. Figure 2&#160;was created with&#160;BioRender.com.

The authors declare no competing interests.

The derivation and analysis of cells from PCa tissue provide a relevant experimental model, particularly considering that available animal models do not fully replicate human prostate anatomy and disease progression37. Chimpanzees&#39; prostate glands are anatomically similar to humans but show a slow, stochastic disease progression, in addition to implying ethical limitations. Dogs differ significantly in prostate structure and pathobiology and develop sporadic, age-related prostate disease. Comm...

Prostate cancer (PCa) is the most frequently diagnosed cancer in men across nearly two-thirds of countries worldwide. In 2022, approximately 1.5 million new cases were reported, resulting in 3,97,000 deaths globally. These figures make PCa the second most common cancer and the fifth leading cause of cancer-related mortality among men1.
PCa is a multifocal and biologically complex disease, marked by substantial intertumoral heterogeneity2,3,4, diverse molecular subtypes, and variable clinical outcomes5,6, all of which significantly influence prognosis and therapeutic response7.
While the critical role of the tumor microenvironment (TME) in prostate tumor initiation and progression is well established8,9, the molecular interactions between tumor cells and the surrounding stromal compartment remain incompletely understood. While it is well established that the prostate tumor microenvironment (TME) plays a critical role in tumor initiation and progression, the molecular interactions between tumor cells and the surrounding stroma remain insufficiently defined. The TME comprises a variety of stromal cells, including those of mesenchymal and immune origin, which become activated in response to tumor-derived signals and subsequently acquire tumor-promoting functions10,11. Among these, cancer-associated fibroblasts (CAFs) often represent the most abundant stromal population. CAFs contribute to extracellular matrix (ECM) remodeling, promote tumor growth, and facilitate cancer cell invasion and migration4,11,12,13. Through secreted factors and direct cell-cell interactions, CAFs also influence proliferation, invasion, and therapy resistance. Notably, CAFs are a heterogeneous population. While generally associated with pro-tumorigenic functions, some subtypes may exert tumor-suppressive roles14,15. Importantly, the absence of unique markers for CAFs poses challenges for their precise identification and the functional dissection of their diverse roles12,16.
A robust approach to investigate the functional crosstalk between CAFs and tumor cells involves isolating primary CAFs and matched normal fibroblasts (NFs), then assessing their respective influence on tumor cell behaviors such as proliferation, migration, colony formation, and anchorage-independent growth17,18,19. This article describes a refined, highly reproducible protocol for isolating CAFs and NFs from radical prostatectomy specimens of high-risk PCa patients. A critical component of this protocol is the accurate identification and dissection of tumor and tumor-free regions by an expert uropathologist, allowing the derivation of CAFs and NFs from the same patient. This paired-sample approach helps control for intertumoral heterogeneity and individual patient-specific variables that can influence fibroblast phenotypes20,21,22. To ensure sufficient tumor tissue is available, we focus on specimens from patients with a Gleason score &#8805;7.
Reliable phenotypic characterization of the isolated fibroblasts requires the use of multiple markers to distinguish them from other cell types, particularly epithelial cells, which may share overlapping marker expression16. As primary PCa fibroblasts typically undergo senescence after 10-15 passages, complicating long-term analyses, we also present a protocol for cell immortalization via stable expression of the human telomerase reverse transcriptase (hTERT)23.
To explore the functional effects of CAFs and NFs on tumor cells, we tested and optimized several experimental strategies. Given that CAFs exert their influence through both secreted factors and direct cell-cell contact24, two complementary assay systems are developed: (1) treatment of cancer cells with fibroblast-conditioned medium (CM), and (2) direct co-culture of fibroblasts and tumor cells. These assays evaluate key cancer cell properties, including proliferation, anchorage-independent growth, and migration. To standardize the influence of secreted factors, conditioned media from CAFs and NFs were concentrated and quantified (conCM) based on total protein content.
Demonstrating significant functional effects through these assays can support the identification of critical mediators of CAF-driven tumorigenesis, offering potential targets for therapeutic intervention.

<table><tbody><tr><td>12 well plate</td><td>Sarstedt</td><td>833921</td><td></td></tr><tr><td>24 well plate</td><td>Sarstedt</td><td>833922</td><td></td></tr><tr><td>6 well plate</td><td>Sarstedt</td><td>833920</td><td></td></tr><tr><td>96 well plate</td><td>Sarstedt</td><td>833924</td><td></td></tr><tr><td>Anti-GAPDH Mouse mAb (6C5)</td><td>Sigma Aldrich</td><td>CB1001</td><td>Source: Mouse; Dilution for WB: 1:1000</td></tr><tr><td>aSMA</td><td>Sigma Aldrich</td><td>A5228&amp;nbsp;</td><td>Source: Mouse; Dilution for WB: 1:3000</td></tr><tr><td>Caveolin</td><td>Cell Signalling Technology</td><td>CST3267</td><td>Source: Rabbit; Dilution for WB: 1:1000</td></tr><tr><td>CD31-VioBlue</td><td>Milteny Biotec</td><td>130-119-980</td><td>Flow Cytometry Ab, Endothelial cell marker</td></tr><tr><td>CD326-PE (EpCAM)</td><td>Milteny Biotec</td><td>130113-826</td><td>Flow Cytometry Ab, Epithelial cell marker</td></tr><tr><td>CD45-FITC</td><td>Milteny Biotec</td><td>130-110769</td><td>Flow Cytometry Ab, Pan leukocyte marker</td></tr><tr><td>CD90-APC</td><td>Milteny Biotec</td><td>130-114-903</td><td>Flow Cytometry Ab, Fibroblast marker</td></tr><tr><td>Collagen VI (COL6A1)</td><td>Abcam</td><td>ab182744</td><td>Source: Rabbit; Dilution for WB: 1:1000</td></tr><tr><td>Conical tube 15 mL</td><td>Sarstedt</td><td>62554502</td><td></td></tr><tr><td>Conical tube 50 mL</td><td>Sarstedt</td><td>62547254</td><td></td></tr><tr><td>Cryopure 2 mL microtubes</td><td>Sarstedt</td><td>72380992</td><td></td></tr><tr><td>Culture plate 100 mm diameter&amp;nbsp;</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Culture plate 100 mm diameter&amp;nbsp;</td><td>Corning</td><td>353025</td><td></td></tr><tr><td>Culture plate 60 mm diameter&amp;nbsp;</td><td>Corning</td><td>353002</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Aldirch</td><td>D8418</td><td>Use in a Biological hood</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Gibco</td><td>11965092</td><td>Store at 4 &amp;deg;C, use in a Biological hood</td></tr><tr><td>FAP</td><td>Abcam</td><td>ab53066</td><td>Source: Rabbit; Dilution for WB: 1:500</td></tr><tr><td>FcR Blocking Reagent, human</td><td>Milteny Biotec</td><td>130-059-901</td><td>Fc Blocker</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000044</td><td>Store at 4 &amp;deg;C, use in a Biological hood</td></tr><tr><td>Filtered tips 10 &amp;micro;L</td><td>Sarstedt</td><td>703010255</td><td></td></tr><tr><td>Filtered tips 100 &amp;micro;L</td><td>Sarstedt</td><td>703030255</td><td></td></tr><tr><td>Filtered tips 1000 &amp;micro;L</td><td>Sarstedt</td><td>703060255</td><td></td></tr><tr><td>Filtered tips 20 &amp;micro;L</td><td>Sarstedt</td><td>703030265</td><td></td></tr><tr><td>Filtered tips 200 &amp;micro;L</td><td>Sarstedt</td><td>703031255</td><td></td></tr><tr><td>Filtropur S Plus 0,20 &amp;micro;m&amp;nbsp;</td><td>Sarstedt</td><td>831826102</td><td></td></tr><tr><td>Forcep</td><td>2Biol</td><td>1102412</td><td></td></tr><tr><td>FSP1/S100A4</td><td>Cell Signalling Technology</td><td>D9F9D (13018)</td><td>Source: Rabbit; Dilution for WB: 1:2000</td></tr><tr><td>IL6</td><td>Abclonal technology</td><td>A0286</td><td>Source: Rabbit; Dilution for WB: 1:1000</td></tr><tr><td>ImageJ</td><td>National Inst. of Health</td><td>version 1.53c</td><td>Software for image analysis</td></tr><tr><td>incucyte imagelock 96-well plate</td><td>Sartorious</td><td>BA-04857</td><td></td></tr><tr><td>Incucyte&amp;reg; SX5</td><td>Sartorious</td><td>SX5</td><td>Live cell analysis instrument&amp;nbsp;</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>11668019</td><td>cation lipid-mediated transfection reagent</td></tr><tr><td>MACS&amp;reg; Tissue Storage Solution</td><td>Milteny Biotec</td><td>130-100-008</td><td></td></tr><tr><td>Microplate reader (600 nm absorbance)</td><td>Promega</td><td>GM3500</td><td></td></tr><tr><td>Microtubes 1.5 mL&amp;nbsp;</td><td>Sarstedt</td><td>7269001</td><td></td></tr><tr><td>Ncad</td><td>Abcam</td><td>ab18203</td><td>Source: Rabbit; Dilution for WB: 1:1000</td></tr><tr><td>Nitro Blue Tetrazolium&amp;nbsp;</td><td>Sigma Aldirch</td><td>n5514</td><td></td></tr><tr><td>Parafilm&amp;nbsp;</td><td>Sigma Aldirch</td><td>HS234526B</td><td></td></tr><tr><td>pBABE-puro-hTERT</td><td>Addgene</td><td>1771</td><td>Store at -20 &amp;deg;C, use in a Biological hood</td></tr><tr><td>pCL-Ampho</td><td>Novus Biological&amp;nbsp;</td><td>NBP2-29541</td><td>Store at -20 &amp;deg;C, use in a Biological hood</td></tr><tr><td>PDGFRB</td><td>Santa Cruz</td><td>sc-432</td><td>Source: Rabbit; Dilution for WB: 1:1000</td></tr><tr><td>Penicillin-Streptomycin (PS, 10,000 &amp;mu;g/mL)&amp;nbsp;</td><td>Gibco</td><td>15140122</td><td>Store at 4 &amp;deg;C, use in a Biological hood</td></tr><tr><td>Phase contrast microscope&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td>BX42</td><td></td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;(PBS)</td><td>Homemade</td><td></td><td>NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 10 mM;KH2PO4, 1.8 mM. Autoclave and Store at RT</td></tr><tr><td>Poly-L-lysine solution</td><td>Sigma Aldirch</td><td>P8920</td><td>Store at 4 &amp;deg;C, use in a Biological hood</td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma Aldirch</td><td>P8833</td><td></td></tr><tr><td>Secura Semi Micro Balance</td><td>Sartorius</td><td>SECURA125-1S</td><td></td></tr><tr><td>Serological pipette 10 mL&amp;nbsp;</td><td>Sarstedt</td><td>861254001</td><td></td></tr><tr><td>Serological pipette 2 mL&amp;nbsp;</td><td>Sarstedt</td><td>861252001</td><td></td></tr><tr><td>Serological pipette 25 mL&amp;nbsp;</td><td>Sarstedt</td><td>861256001</td><td></td></tr><tr><td>Serological pipette 5 mL&amp;nbsp;</td><td>Sarstedt</td><td>861253001</td><td></td></tr><tr><td>STAT3</td><td>CST</td><td>9139</td><td>Source: Mouse; Dilution for WB: 1:1000</td></tr><tr><td>STAT3 Y705</td><td>CST</td><td>9131</td><td>Source: Rabbit; Dilution for WB: 1:1000</td></tr><tr><td>stereomicroscope</td><td>Leica</td><td>MZ125</td><td></td></tr><tr><td>Syringe 10 mL</td><td>Pentaferte</td><td>00202260D12</td><td></td></tr><tr><td>TermoMixer</td><td>Eppendorph</td><td>22331</td><td></td></tr><tr><td>Trypsin-EDTA (0.5%), no phenol red</td><td>Gibco</td><td>15400054</td><td></td></tr><tr><td>Vim</td><td>Santa Cruz</td><td>sc-6260</td><td>Source: Mouse; Dilution for WB: 1:2000</td></tr><tr><td>Vivaspin 20 centrifugal concentrator&amp;nbsp;</td><td>Sigma Aldirch</td><td>Z614602</td><td>MWCO 10,000 Da&amp;nbsp;</td></tr><tr><td>Wagner scissors</td><td>2Biol</td><td>14070-12</td><td></td></tr></tbody></table>

isolation, culture, and characterization of prostate cancer-associated fibroblasts

Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts

Tumor stroma can be actively shaped by tumor cells to adopt pro-tumorigenic properties, making it a focal point of cancer research. Among the stromal components, cancer-associated fibroblasts (CAFs) -- often the most abundant cell type in the tumor microenvironment (TME) -- play a pivotal role in promoting cancer progression through their interactions with tumor cells and other stromal components. Elucidating the mechanisms behind these interactions requires robust methods for isolating&#160;and characterizing&#160;CAFs, which can also lay the foundation for developing novel CAF-targeted cancer therapies. This study presents a method for isolating fibroblasts from both the tumor and the adjacent normal regions of radical prostatectomy specimens obtained from prostate cancer (PCa) patients. This approach enables the concurrent isolation of CAFs and their normal counterparts (NFs) from the same individual, providing a paired experimental system. Detailed protocols are provided for the maintenance of primary CAFs and NFs for downstream in vitro analyses, as well as for their immortalization to facilitate long-term studies. We also describe functional assays designed to compare the effects of CAFs and NFs on cancer cell behaviors, including proliferation, migration, and anchorage-independent growth. Two complementary approaches are detailed: treatment of cancer cells with fibroblast-derived conditioned media (CM), and direct co-culture of fibroblasts with tumor cells. Together, these methodologies represent&#160;a comprehensive toolkit for investigating the dynamic interplay between CAFs and tumor cells, not only in PCa but potentially across a range of human cancers. Moreover, molecular characterization of these interactions may reveal key mediators of CAF-driven oncogenesis, offering promising targets for therapeutic intervention.

CAF and NF pairs from 7 high-grade PCa patients were successfully isolated with this protocol. Patients' age at surgery and Gleason scores are summarized in Table 3. To evaluate the purity of the derived fibroblast populations, passage two primary CAFs and NFs were detached and stained with the indicated fluorescent antibodies or with propidium iodide to assess apoptosis. Representative results from the subsequent FACS analysis are shown in Figure 2A...

This protocol provides a comprehensive procedure to isolate, expand, and immortalize fibroblasts from radical prostatectomies. Moreover, it describes assays developed to assess the functional effects of fibroblast-tumor cell crosstalk, taking advantage of both treatment with conditioned medium and co-culturing.

Tumor stroma can be actively shaped by tumor cells to adopt pro-tumorigenic properties, making it a focal point of cancer research. Among the stromal ...

We aim to understand the mechanism by which Cancer-Associated Fibroblast contribute to tumor progression in prostate cancer with the goal to neutralize this crosstalk as a therapeutic strategy. Distinct populations of CAFs with different properties can be assessed in 3D organoids obtained using tumor and stromal cells upon various manipulations. Our protocol allows to reliably generate CAFs for these purposes.Main challenges are the reliable isolation of tumor and normal cells from the same patients'microenvironments, heterogeneity, absence of specific CAF markers, CAFs'plasticity, and the limited in vitro models recapitulating TME complexity. With similar methods, we characterized the fundamental pro-tumor role of the transcription factor STAT3 and its target genes in murine breast cancer CAFs, demonstrating their efficacy as therapeutic targets. An optimal isolation of fibroblasts from the tumor and the adjacent normal regions of radical prostatectomy specimens obtained from prostate cancer patients using small amount of the tissue.To begin, weigh the tissue samples on the balance on day one for fibroblast isolation. Using sterile forceps, transfer the tissue into six centimeter dishes. Wash the tissue twice in four milliliters of ice cold PBS and twice in four milliliters of ice cold complete DMEM.Now, transfer the tissue into a six centimeter plate on ice. Add one milliliter of antibiotic supplemented DMEM to the plate. Then use scissors or blades to mince the tissue into fragments smaller than one square millimeter.Transfer the minced tissue to a 15 milliliter conical tube containing five milliliters of ice cold complete DMEM. Then centrifuge the suspension at 754 g for five minutes at four degrees Celsius. Using a 10 milliliter pipette, remove the supernatant.Resuspend the pellet in five milliliters of ice cold complete DMEM and centrifuge again. After removing the supernatant, resuspend the pellet in collagenase II solution. Transfer the suspension into 1.5 milliliter microtubes.Seal the microtubes with paraffin film. Then place them at 37 degrees Celsius for overnight digestion with continuous rocking for eight to 12 hours. The next day, transfer the digested samples to 15 milliliter conical tubes.Pipette five milliliters of ice cold complete DMEM to inactivate collagenase. Then centrifuge the suspension at 754 g for five minutes at four degrees Celsius. Resuspend the pellet in one milliliter of 0.05%trypsin-EDTA after pipetting out the supernatant.Incubate for five minutes at 37 degrees Celsius with occasional shaking. Next, pipette one milliliter of freshly prepared DNase I solution to the samples and mix well. After centrifuging and removing the supernatant, resuspend the pellet in five milliliters of cold complete DMEM and centrifuge again.Resuspend the resulting cells in complete DMEM supplemented with 20%fetal bovine serum. Plate the cells and incubate at 37 degrees Celsius with 5%carbon dioxide and 95%humidity for at least three days. After three days, examine the cells for morphology and viability.Once the cells reach confluency, passage them into a 10 centimeter dish containing complete DMEM with 20%fetal bovine serum. Plate cancer-associated fibroblasts or normal fibroblasts at 70%confluency in 12 well plates. On the following day, add 0.45 grams of low melting point agar to 12.5 milliliters of PBS in a 50 milliliter conical tube under sterile conditions.Dissolve the mixture using a microwave oven. Cool the agar solution down to 37 degrees Celsius. Then dilute in a one to four ratio with pre-warmed complete DMEM to prepare a working solution.Aspirate the medium from the 12 well plate. Pipette 500 microliters of the working agar solution into each well. Incubate for 20 minutes at four degrees Celsius to allow for agar solidification.Meanwhile, keep the remaining agar solution at 37 degrees Celsius. Trypsinize the tumor cells and prepare a cell suspension of 20, 000 cells per milliliter. Mix equal volumes of the tumor cell suspension and the agar solution to obtain a final volume of seven milliliters, accounting for three technical replicates per each condition.Pipette up and down multiple times to ensure even mixing. Then dispense 500 microliters of this mixture containing approximately 5, 000 cells on top of the solidified base layer in each well. After letting the agar solidify for 20 minutes at four degrees Celsius, add one milliliter of complete DMEM to each well.Change the medium every other day by aspirating gently from the edge of the well and adding fresh medium to the center. Incubate until colonies become easily visible. Once colonies are visible, discard the culture medium.Then stain the colonies with 200 microliters of Nitro blue tetrazolium chloride solution. Incubate overnight at 37 degrees Celsius in a humidified incubator. The next day, discard the staining solution.Acquire images of stained colonies using a stereo microscope. Pure fibroblasts were successfully isolated. In many cases, particularly at early passages, cancer-associated fibroblasts displayed a more spindle-like morphology compared to normal fibroblasts.Quantitative analysis confirmed that proliferation of DU145 cells treated with cancer-associated fibroblast conditioned media was significantly higher over 120 hours than those treated with normal fibroblast conditioned media or left untreated. DU145 cells co-cultured directly with cancer-associated fibroblasts and normal fibroblasts also showed increased proliferation over 96 hours relative to the controls. Corresponding growth curves demonstrated that co-culture with cancer-associated and normal fibroblasts resulted in a similar increase in DU145 proliferation over time.The effect of conditioned media on DU145 proliferation varied across cancer-associated fibroblast and normal fibroblast pairs with early passage pairs showing significantly stronger effects than later ones. In soft agar colony formation assays, both cancer-associated and normal fibroblasts increased the number and size of DU145 colonies compared to controls, indicating enhanced anchorage-independent growth. Detached fibroblast layers formed due to technical issues prevented colony formation in some replicates.Conditioned media alone did not promote anchorage-independent colony formation in DU145 cells.

isolation-culture-characterization-prostate-cancer-associated

Research

JoVE Journal

Cancer Research

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 PDGFRα as a surface marker.

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

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Three-dimensional Co-culture Model for Tumor-stromal Interaction

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.

 Murine Dermal Fibroblast Isolation by FACS

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

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.

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

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).

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

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.

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

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...

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

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Learn to isolate, culture, and characterize prostate cancer-associated fibroblasts from radical prostatectomy specimens. This method enables reliable generation of CAFs for studying tumor-stromal interactions and therapeutic targets.

Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts

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