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      Towards organoid culture without Matrigel

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          Abstract

          Organoids—cellular aggregates derived from stem or progenitor cells that recapitulate organ function in miniature—are of growing interest in developmental biology and medicine. Organoids have been developed for organs and tissues such as the liver, gut, brain, and pancreas; they are used as organ surrogates to study a wide range of questions in basic and developmental biology, genetic disorders, and therapies. However, many organoids reported to date have been cultured in Matrigel, which is prepared from the secretion of Engelbreth-Holm-Swarm mouse sarcoma cells; Matrigel is complex and poorly defined. This complexity makes it difficult to elucidate Matrigel-specific factors governing organoid development. In this review, we discuss promising Matrigel-free methods for the generation and maintenance of organoids that use decellularized extracellular matrix (ECM), synthetic hydrogels, or gel-forming recombinant proteins.

          Abstract

          Kozlowski, Crook, and Ku review current state-of-the-art techniques in culturing and manipulating organoids without the use of EHS-tumor-derived matrix, the most popular of which is Matrigel by Corning. They cover organoid-based applications of different matrix types, ranging from natural sources (decellularized ECM, purified ECM proteins, polysaccharides), synthetic sources (e.g. PEG-based), and recombinant peptide-based systems.

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          Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.

          The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
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            Matrix elasticity directs stem cell lineage specification.

            Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.
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              Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

              We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                mark.t.kozlowski4.civ@army.mil
                Journal
                Commun Biol
                Commun Biol
                Communications Biology
                Nature Publishing Group UK (London )
                2399-3642
                10 December 2021
                10 December 2021
                2021
                : 4
                : 1387
                Affiliations
                [1 ]DEVCOM US Army Research Laboratory, Weapons and Materials Research Directorate, Science of Extreme Materials Division, Polymers Branch, 6300 Rodman Rd. Building 4600, Aberdeen Proving Ground, Aberdeen, MD 21005 USA
                [2 ]GRID grid.410425.6, ISNI 0000 0004 0421 8357, Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, , City of Hope National Medical Center, ; 1500 Duarte Rd., Duarte, CA 91010 USA
                [3 ]GRID grid.410425.6, ISNI 0000 0004 0421 8357, Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, ; 1500 Duarte Rd., Duarte, CA 91010 USA
                [4 ]GRID grid.410425.6, ISNI 0000 0004 0421 8357, Present Address: Department of Medical Oncology and Therapeutics Research, , City of Hope National Medical Center, ; 1500 Duarte Rd., Duarte, CA 91010 USA
                Author information
                http://orcid.org/0000-0003-4714-8697
                http://orcid.org/0000-0003-2065-9817
                http://orcid.org/0000-0001-5662-9893
                Article
                2910
                10.1038/s42003-021-02910-8
                8664924
                34893703
                cb86d73a-51e5-4d99-81f2-917465031b32
                © This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2021

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 8 October 2020
                : 24 November 2021
                Funding
                Funded by: FundRef https://doi.org/10.13039/100000005, U.S. Department of Defense (United States Department of Defense);
                Award ID: NDSEG
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000002, U.S. Department of Health & Human Services | National Institutes of Health (NIH);
                Award ID: R01DK099734
                Award Recipient :
                Categories
                Review Article
                Custom metadata
                © The Author(s) 2021

                biomaterials,stem-cell differentiation,biological models

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