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style="background-image:url(https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2023/03/iStock-1390650720_connections.jpg)"> </section> <div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-18470 post type-post status-publish format-standard has-post-thumbnail category-cell-therapy category-general category-gibco tag-gibco tag-organoids tag-spheroids tag-tumoroids entry" aria-label="How 3D Cell Culture Models Work"><header class="entry-header"><h1 class="entry-title">How 3D Cell Culture Models Work</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"> 09.12.2024 </span> </header><div class="entry-content"><p>The term “3D model” might make one think of graphic design or hand-painted miniatures, but in cell culture, it’s something truly remarkable. In contrast to traditional 2D cell culture which relies on cells grown in a single plane, 3D cell culture models are clusters of cells that can grow, differentiate, and organize into a complex structure that exhibits similar behavior and functions as the tissues from which they were cultured. The more representative cellular environment that 3D cell models provide results in an invaluable tool to explore areas of research that cannot be achieved using traditional 2D models. These models provide an ideal cellular environment for a variety of applications such as tissue engineering, cell therapy, disease modeling, tumor biology, drug discovery, and personalized medicine.</p> <h2>Kinds of 3D Cell Culture Model</h2> <p>3D cell culture models can be derived from immortalized cell lines, primary or patient-derived cells, or from stem cells<sup>1</sup>. They can grow, proliferate, and differentiate and spatially arrange into different cell types. What they have in common is that they are capable of self-renewal in the right culture media and behave, in many ways, like ordinary tissues and organs. Depending on their origin, these self-aggregating 3D cell culture models fall into three main categories:</p> <ul> <li><strong>Spheroids </strong>are 3D cellular aggregates derived from immortalized cell lines. They are composed of one or more cell types that grow and proliferate, and may exhibit enhanced physiological responses, but do not undergo differentiation or self-organization.</li> <li><strong>Organoids</strong> are 3D structures derived from pluripotent stem cells (PSCs), neonatal tissue stem cells, or adult stem cells. In organoids, cells spontaneously self-organize into properly differentiated functional cell types and progenitors that resemble their <em>in vivo</em> counterparts and recapitulate at least some function of the organ<sup>2</sup>. Organoids assemble and organize themselves, capture the complexities of their derived organs, display representative cellular polarity, and recapitulate proper cellular spatial architecture.</li> <li><strong>Tumoroids </strong>are patient-derived cancer cells grown as three-dimensional, self-organized, multicellular structures. These work best for studying complex, solid tumors which require complex and specific media systems.</li> </ul> <p><img decoding="async" class="alignnone wp-image-18513 size-large" src="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/3D_blog-pic-1024x825.png" alt="Spheroids, Organoids and Tumoroids" width="760" height="612" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/3D_blog-pic-1024x825.png 1024w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/3D_blog-pic-300x242.png 300w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/3D_blog-pic-768x619.png 768w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/3D_blog-pic.png 1176w" sizes="(max-width: 760px) 100vw, 760px" /></p> <p>All three of these models offer key advantages over relying solely on traditional i<em>n vitro</em> or <em>in vivo</em> systems. Immortalized cell lines in petri dishes might be far less challenging to grow and maintain, but they have limited physiological and clinical relevance. Such isolated systems cannot emulate more complex intercellular and organ-level interactions. Conversely, trials in whole organisms (whether laboratory or clinical, rodent or human) are large, expensive, time-consuming affairs that require complex ethical approval processes to even attempt, making them best saved for when other kinds of studies have already yielded positive results. 3D cell culture models are at the midpoint of these extremes: much less expensive and more efficient than clinical trials, but far closer to working with whole, living organisms than ordinary cell lines. Using 3D cell culture models enables researchers to use relevant <em>in vitro </em>studies to collect more information and make their future <em>in vivo </em>studies more focused and effective.</p> <h2>Steps to Success</h2> <p>Creating and using a 3D cell culture model has five phases, and Gibco has numerous tools, reagents, and consumables for every step of the process.</p> <h3><strong>Culture</strong></h3> <p>First, the researchers must choose, harvest, grow, count, and establish their cell sources. Different types and combinations of cells can be used for different research goals. The chosen cells dictate the biology, complexity, and growth conditions of the resulting culture.</p> <h3><strong>Create</strong></h3> <p>Once the cells are growing, it is time to form them into their respective 3D cell model. This involves collecting cells, this time from the newly established culture, and growing them in specific media, cell culture plastics, or extracellular matrices that encourage cell clustering. Various growth factors and proteins may be required in their media for them to grow and develop properly.</p> <h3><strong>Characterize</strong></h3> <p>The next step is demonstrating 3D cell model health and relevance. A cell culture, 3D or otherwise, cannot serve its intended purpose if the cells in it are nonviable, and 3D cell cultures additionally must form their desired 3D structures to be useful. Various plate-based or image-based assays can be employed to determine relative cell health and distinguish live from dead cells in a 3D cell culture. For tumoroids, genetic assays such as next generation sequencing or whole transcriptome RNASeq are necessary to make sure that the tumoroid exhibits the same mutations and gene expression patterns as the original donor sample. Deviation of these tumor characteristics limits the relevance of a developed tumoroid model.</p> <h3><strong>Engineer</strong></h3> <p>At this point in the process, one has already created a 3D cell culture model, but it might not be the exact model needed to answer one’s research questions. Standard genetic engineering techniques, including CRISPR-Cas9 and lentiviral vectors, are useful here, to insert or delete specific mutations whose effects researchers want to test or to generate a stable reporter cell line. Gibco’s suite of electroporation devices and lentiviral production systems are necessary to aid cells in taking in foreign material, which can make this a smooth and relatively simple process.</p> <h3><strong>Analyze</strong></h3> <p>Once one’s 3D model is healthy, relevant, and possibly modified, it is time to test one’s creation in the presence of a compound or drug and measure the effects. Whether the goal is to check whether a new compound can kill cancer tumoroids, check whether a genetic change leads to new protein expression, or something else, Gibco has the tools to make it work. The Gibco<sup>TM</sup> portfolio of 3D cell culture products is manufactured in <a href="https://ispe.org/initiatives/regulatory-resources/gmp">GMP-compliant settings</a>, safety tested, and backed by regulatory documentation to support the whole sequence of 3D cell culture, from collecting starting material all the way to measuring experimental results. <a href="https://www.thermofisher.com/ca/en/home/life-science/cell-culture/organoids-spheroids-3d-cell-culture.html?icid=bid_clb_can_r01_co_cp1610_pjt12011_bidclb3d1_0so_blg_op_awa_kt_siz_3dcellculmod1">Gibco offers instrumentation, software, consumables, and dedicated testing, regulatory, and quality control support for every step in the 3D cell culture process</a>. Gibco also offers <a href="https://www.thermofisher.com/ca/en/home/life-science/cell-culture/organoids-spheroids-3d-cell-culture.html?icid=bid_clb_can_r01_co_cp1610_pjt12011_bidclb3d1_0so_blg_op_awa_kt_siz_3dcellculmod2">protocols</a>, <a href="https://www.thermofisher.com/ca/en/home/global/forms/life-science/download-3d-cell-culture-analysis-handbook.html?icid=bid_clb_can_r01_co_cp1610_pjt12011_bidclb3d1_0so_blg_op_awa_kt_siz_3dcellculmod3">handbooks</a>, <a href="https://www.thermofisher.com/account-center/cloud-signin-identifier.html?icid=bid_clb_can_r01_co_cp1610_pjt12011_bidclb3d1_0so_blg_op_awa_kt_siz_3dcellculmod4">e-learning resources</a>, and <a href="https://www.thermofisher.com/ca/en/home/life-science/cell-culture/mammalian-cell-culture/cancer-cell-culture/tumoroid-culture.html?icid=bid_clb_can_r01_co_cp1610_pjt12011_bidclb3d1_0so_blg_op_awa_kt_siz_3dcellculmod5">workflows</a> for spheroid, organoid, and tumoroid research to guide new researchers on the way to becoming experts in this challenging field.</p> <p>Have a look at our catalog and web resources today to find out how to get started with 3D cell culture models.</p> <ol> <li>Fatehullah A, SH Tan, N Barker (2016) Organoids as an in vitro model of human development and disease. <em>Nat. Cell Biol.</em> 18(3):246–254.</li> <li>Huch M, B-K Koo (2015) Modeling mouse and human development using organoid cultures. <em>Dev. Camb. Engl.</em> 142(18):3113–3125.</li> </ol> <p>© 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.</p> <p> </p> <h5><strong>Related Content:</strong></h5> <p><a href="https://www.thermofisher.com/us/en/home/brands/gibco.html?cid=bid_clb_gbo_r01_co_cp1610_pjt12011_ext6741_0wm_tfs_xl_awa_og_s00_gwg_blog_2024" target="_blank" rel="noopener">Gibco Brand</a></p> <p><a href="https://www.thermofisher.com/blog/behindthebench/co-culturing-patient-derived-cancer-organoids-with-fibroblasts-and-endothelial-cells/?cid=bid_clb_gbo_r02_us_cp1610_pjt12011_col026956_0wm_tfs_xl_awa_og_s00_GwG_ipsc_blog" target="_blank" rel="noopener">Co-culturing patient-derived cancer organoids with fibroblasts and endothelial cells</a></p> <p><a href="https://www.thermofisher.com/blog/behindthebench/cancer-organoids-vs-cancer-spheroids/" target="_blank" rel="noopener">Cancer organoids vs cancer spheroids</a></p> <p><a 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