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cells</span></div></div></header><div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-18375 post type-post status-publish format-standard has-post-thumbnail category-cancer-research category-cell-therapy category-general tag-cancer-cell-line tag-cancer-models tag-cancer-organoid tag-co-culture tag-popular tag-tumor-microenvironment tag-tumor-model tag-tumoroid entry" aria-label="Co-culturing patient-derived cancer organoids with fibroblasts and endothelial cells"><header class="entry-header"><h1 class="entry-title">Co-culturing patient-derived cancer organoids with fibroblasts and endothelial cells</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/behind-the-bench-staff/" title="Posts by Behind The Bench Staff" rel="author">Behind The Bench Staff</a> </span> <span class="post-info-date"> 06.06.2024 </span> </header><div class="entry-content"><p>In cancer research, the development of accurate and representative cell models is crucial for understanding tumor biology and improving therapeutic strategies. Traditional immortalized cell lines have limitations in mimicking the complexity of patient tumors, including real-life gene expression patterns, and clinically observed mutations. In contrast, patient-derived cancer organoids (also known as tumoroids), which are obtained directly from patient samples and grown in 3D culture systems using specialized serum-free media such as <a href="https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/cancer-cell-culture/tumoroid-culture/tumoroid-medium.html?cid=bid_clb_can_r01_co_cp1610_pjt12005_col28408_0so_blg_il_cdn_an_s00_oncoproblogs" target="_blank" rel="noopener">OncoPro Tumoroid Culture Medium</a>, more accurately reflect the biology and heterogeneity of patient cancers compared to immortalized cell lines. Most importantly, tumoroids retain the genetic and phenotypic characteristics of the original tumor. An important piece of studying tumoroid biology is understanding how these cancer models interact with other cell populations that are native in humans. To do this, we establish tumoroid co-cultures in which tumoroids and other cell types of interest are combined and cultured together in the same dish or flask.</p> <div id="attachment_18380" style="width: 299px" class="wp-caption alignright"><img decoding="async" aria-describedby="caption-attachment-18380" class=" wp-image-18380" src="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-1-coculture_updated.png" alt="Cancer organoid co-culture" width="289" height="298" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-1-coculture_updated.png 586w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-1-coculture_updated-291x300.png 291w" sizes="(max-width: 289px) 100vw, 289px" /><p id="caption-attachment-18380" class="wp-caption-text"><em><strong>Figure 1.</strong> <strong>Tumoroid co-culture incorporating fibroblasts and endothelial cells.</strong></em><br /><em>HuCo021320 colorectal tumoroids were co-cultured with BJ skin fibroblasts (green) and HMEC-1 endothelial cells (red). Fibroblasts and tumoroid cells were mixed 1:1, and endothelial cells were added at 25% of the (tumoroid + fibroblast) population. Structures were imaged on a CellInsight CX7 LZR microscope at 10 days post-seeding. Scale bar = 100 µm.</em></p></div> <h2>The tumor microenvironment and fibroblasts</h2> <p>The tumor microenvironment, consisting of various cell types and extracellular components, plays a crucial role in tumor progression and response to therapies. It influences cancer cell behavior, angiogenesis, immune response, and drug resistance. Mimicking the unique tumor microenvironment within cancer cell cultures is essential for understanding these complex interactions and developing effective treatments. Key components of the microenvironment are fibroblasts and endothelial cells. Fibroblasts provide structural support, produce extracellular matrix components, and secrete growth factors that promote tumor growth. Endothelial cells form the blood vessels that supply nutrients and oxygen to the tumor. Establishing a co-culture that includes these cell types and tumoroid cells enables the study of their cell-to-cell interactions and contributions to tumor behavior (Figure 1).</p> <h2>Tumoroid co-culture design</h2> <p>When designing co-culture experiments, there are several key considerations to keep in mind: choice of culture media identification of each cell type, and experiment duration.</p> <h3>Co-culture media selection</h3> <p>For media selection, we recommend initiating the co-culture with cell culture medium that supports the most sensitive cell type to ensure the survival and function of all cells. Another beneficial approach can be to blend the media used to culture each individual cell type. Mixing media at a 1:1 ratio (or 1:1:1, depending on the number of cell types being co-cultured) is a good starting point to provide a balanced environment that supports the growth and interaction of all co-cultured cells. Further refinement to the media blend can be done depending on the experimental readout of interest.</p> <h3>Cell staining</h3> <p>To enable accurate identification and tracking of different cell types within the co-culture model, cells can be stained with distinct fluorophores for each type prior to combining and culturing them together. Another option is to incorporate cells that endogenously express fluorescent proteins. With either method, the goal is to be able to track the various behaviors and interactions that different cell types may exhibit within the tumoroid co-culture environment.</p> <div id="attachment_18382" style="width: 371px" class="wp-caption alignright"><img decoding="async" aria-describedby="caption-attachment-18382" class=" wp-image-18382" src="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-2-coculture_updated.png" alt="Localization in cancer organoid co-culture" width="361" height="284" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-2-coculture_updated.png 1376w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-2-coculture_updated-300x236.png 300w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-2-coculture_updated-1024x805.png 1024w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/06/Figure-2-coculture_updated-768x604.png 768w" sizes="(max-width: 361px) 100vw, 361px" /><p id="caption-attachment-18382" class="wp-caption-text"><em><strong>Figure 2. Localization of fibroblasts and endothelial cells in co-culture with colorectal tumoroids.</strong></em><br /><em>HuCo021320 colorectal tumoroids were dissociated and combined with primary adult fibroblasts (green) at (A,B) 1:1 or (C,D) 1:0.5 ratios. Dissociated HMEC-1 endothelial cells (red) were added at 25% of the (tumoroid+fibroblast) population. Co-cultures were grown in OncoPro Tumoroid Culture Medium for 10-11 days and then imaged with a CellInsight CX7 LZR microscope or fixed, sectioned, and imaged on an EVOS M7000 microscope. Scale bar = 100 µm on confocal images and 125 µm on sections.</em></p></div> <h3>Monitoring</h3> <p>In many cases, it may be difficult to maintain multiple cell types at steady ratios over long-term culture. Initial pilot experiments in which cultures are routinely monitored, in conjunction with the cell tracking techniques discussed above, will help establish an experimental window where all cells are viable. Depending on the purpose of the experiment, monitoring may include a functional readout (e.g., perfusion in vasculature, fibroblast morphology, cytokine production) to ensure that different cellular compartments are functioning as required.</p> <h2>Tumoroid co-culture in practice</h2> <p>We have performed co-culture experiments using tumoroids, endothelial cell lines, and either fibroblast cell lines or primary adult fibroblasts. To track fibroblasts and endothelial cells, we stained each cell type with <a href="https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/cell-tracking.html?cid=bid_clb_can_r01_co_cp1610_pjt12005_col28408_0so_blg_il_cdn_an_s00_oncoproblogs" target="_blank" rel="noopener">CellTracker fluorescent dyes</a><a href="https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/cell-tracking.html?cid=bid_clb_can_r01_co_cp1610_pjt12005_col28408_0so_blg_il_cdn_an_s00_oncoproblogs"> </a> for 45 minutes in serum-free medium prior to combining them with tumoroid cells. For co-culture, we then dissociated tumoroid cells, fibroblasts, and endothelial cells and combined them at various ratios. We maintained ratios of tumoroid cells to fibroblasts at 1:1 or 1:0.5, with the addition of endothelial cells to account for approximately 25% of the total cell number. For media selection, we choose to co-culture using <a href="https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/cancer-cell-culture/tumoroid-culture/tumoroid-medium.html?cid=bid_clb_can_r01_co_cp1610_pjt12005_col28408_0so_blg_il_cdn_an_s00_oncoproblogs" target="_blank" rel="noopener">OncoPro Tumoroid Culture Medium</a> supplemented with 2% (v/v) <a href="https://www.thermofisher.com/order/catalog/product/A1569601?cid=bid_clb_can_r01_co_cp1610_pjt12005_col28408_0so_blg_il_cdn_an_s00_oncoproblogs" target="_blank" rel="noopener">Geltrex</a>. We suggest monitoring the co-culture for up to 10-11 days post-seeding to allow for tumoroid formation. To determine the distribution of cells within the co-culture models, we utilized confocal microscopy via the <a href="https://www.thermofisher.com/order/catalog/product/HCSDCX7LZRPRO?cid=bid_clb_can_r01_co_cp1610_pjt12005_col28408_0so_blg_il_cdn_an_s00_oncoproblogs" target="_blank" rel="noopener">CellInsight CX7 LZR Pro</a>. These methods allow for detailed visualization and analysis of cell interactions and spatial organization within the co-culture. In our hands, fibroblasts spread on the outer surface of cystic HuCo021320 tumoroids, and HMEC-1 endothelial cells localized to the internal lumen of the tumoroids (Figure 2).</p> <h2>Conclusion</h2> <p>Co-culturing patient-derived tumoroids with fibroblasts and endothelial cells provides a more physiologically relevant model to study the tumor microenvironment. By optimizing co-culture conditions and employing advanced analysis techniques, we can gain valuable insights into tumor biology, drug response, and potential therapeutic strategies.</p> <p>Learn more about tumoroids at <a href="https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/cancer-cell-culture/tumoroid-culture.html?cid=bid_clb_can_r01_co_cp1610_pjt12005_col28408_0so_blg_il_cdn_an_s00_oncoproblogs" target="_blank" rel="noopener">www.thermofisher.com/tumoroid</a></p> <p> </p> <h5>Related Content:</h5> <p><a href="https://www.thermofisher.com/blog/behindthebench/how-3d-cell-culture-models-work/?cid=bid_clb_gbo_r01_co_cp1610_pjt12011_ext6741_0wm_tfs_xl_awa_og_s00_gwg_blog_2024" target="_blank" rel="noopener">How 3D Cell Culture Models Work</a></p> <p class="selectionShareable"><a href="https://www.thermofisher.com/blog/behindthebench/cancer-organoids-vs-cancer-spheroids/" target="_blank" rel="noopener">Cancer organoids vs cancer spheroids</a></p> <aside class="mashsb-container mashsb-main "><div class="mashsb_above_buttons">Share this article </div><div class="mashsb-box"><div class="mashsb-buttons"><a class="mashicon-facebook mash-large mashsb-noshadow" href="https://www.facebook.com/sharer.php?u=https%3A%2F%2Fwww.thermofisher.com%2Fblog%2Fbehindthebench%2Fco-culturing-patient-derived-cancer-organoids-with-fibroblasts-and-endothelial-cells%2F" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Facebook</span></a><a class="mashicon-linkedin mash-large mashsb-noshadow" href="https://www.linkedin.com/shareArticle?trk=Co-culturing+patient-derived+cancer+organoids+with+fibroblasts+and+endothelial+cells&url=https%3A%2F%2Fwww.thermofisher.com%2Fblog%2Fbehindthebench%2Fco-culturing-patient-derived-cancer-organoids-with-fibroblasts-and-endothelial-cells%2F" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Linkedin</span></a><a class="mashicon-twitter mash-large mashsb-noshadow" href="https://twitter.com/intent/tweet?text=Co-culturing%20patient-derived%20cancer%20organoids%20with%20fibroblasts%20and%20endothelial%20cells&url=https://www.thermofisher.com/blog/behindthebench/?p=18375&via=thermofisher" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Twitter</span></a><a class="mashicon-mail mash-large mashsb-noshadow" href="mailto:?subject=Check%20out%20this%20article%20from%20Accelerating%20Science&body=Check%20out%20this%20article%20I%20just%20read%3Ahttps%3A%2F%2Fwww.thermofisher.com%2Fblog%2Fbehindthebench%2Fco-culturing-patient-derived-cancer-organoids-with-fibroblasts-and-endothelial-cells%2F" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Mail</span></a><div class="onoffswitch2 mash-large mashsb-noshadow" style="display:none"></div></div> </div> <div style="clear:both"></div><div class="mashsb-toggle-container"></div></aside> </div></article><div class="adjacent-entry-pagination pagination"><div class="pagination-previous alignleft"><i class="fa fa-icon fa-chevron-left"></i> <a href="https://www.thermofisher.com/blog/behindthebench/application-of-raman-spectroscopy-as-process-analytical-technology-pat-in-biopharmaceutical-manufacturing/" rel="prev">Application of Raman Spectroscopy as Process Analytical Technology (PAT) in Biopharmaceutical Manufacturing</a></div><div class="pagination-next alignright"><a href="https://www.thermofisher.com/blog/behindthebench/the-essential-role-of-checkweighers-in-pharmaceutical-production/" rel="next">The Essential Role of Checkweighers in Pharmaceutical Production</a> <i class="fa fa-icon fa-chevron-right"></i></div></div></main></div></div><footer class="site-footer"><div class="wrap"><div class="creds"><p class="footer-links"><a href="https://corporate.thermofisher.com/en/footer/privacy-statement.html" target="_blank" rel="noopener noreferrer">Privacy Statement</a><a href="https://corporate.thermofisher.com/en/footer/terms-and-conditions.html" target="_blank" rel="noopener noreferrer">Terms & Conditions</a><a href="/blog/locations/">Locations</a><a href="/blog/sitemap/">Sitemap</a></p><p>© 2024 <a href="https://www.thermofisher.com">Thermo Fisher Scientific</a>. 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