{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T09:46:23Z","timestamp":1753868783211,"version":"3.41.2"},"reference-count":7,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2023,12,6]],"date-time":"2023-12-06T00:00:00Z","timestamp":1701820800000},"content-version":"vor","delay-in-days":5,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2023,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>For the past decade, three\u2010dimensional (3D) culture models have been emerging as powerful tools in translational research to overcome the limitations of two\u2010dimensional cell culture models. Thanks to their ability to recapitulate the phenotypic and molecular heterogeneity found in numerous organs, organoids have been used to model a broad range of tumors, such as colorectal cancer. Several approaches to generate organoids exist, with protocols using either pluripotent stem cells, embryonic stem cells, or organ\u2010restricted adult stem cells found in primary tissues, such as surgical resections as starting material. The latter, so\u2010called patient\u2010derived organoids (PDOs), have shown their robustness in predicting patient drug responses compared to other models. Because of their origin, PDOs are natural offspring of the patient tumor or healthy surrounding tissue, and therefore, have been increasingly used to develop targeted drugs and personalized therapies. Here, we present a new protocol to generate patient\u2010derived colon organoids (PDCOs) from tumor and healthy tissue biopsies. We emphasize budget\u2010friendly and reproducible techniques, which are often limiting factors in this line of research that restrict the development of this 3D\u2010culture model to a small number of laboratories worldwide. Accordingly, we describe efficient and cost\u2010effective techniques to achieve immunoblot and high\u2010resolution microscopy on PDCOs. Finally, a novel strategy of lentiviral transduction of PDCOs, which could be applied to all organoid models, is detailed in this article. \u00a9 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Establishment of PDCOs from biopsies<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Long\u2010term maintenance and expansion of PDCOs in BME domes<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Cryopreservation and thawing of PDCOs<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Lentiviral transduction of PDCOs<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Immunoblot and evaluation of variability between donors<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: Immunofluorescence labeling and high\u2010resolution microscopy of PDCOs<\/jats:p><jats:p><jats:bold>Basic Protocol 7<\/jats:bold>: Transcriptomic analyses of PDCOs by RT\u2010qPCR<\/jats:p>","DOI":"10.1002\/cpz1.943","type":"journal-article","created":{"date-parts":[[2023,12,7]],"date-time":"2023-12-07T23:52:06Z","timestamp":1701993126000},"source":"Crossref","is-referenced-by-count":0,"title":["Budget\u2010Friendly Generation, Biochemical Analyses, and Lentiviral Transduction of Patient\u2010Derived Colon Organoids"],"prefix":"10.1002","volume":"3","author":[{"given":"Emilie","family":"Rigaux","sequence":"first","affiliation":[{"name":"University of Namur, URBC \u2013 Unit of Research in Cell Biology  Namur Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jia\u2010Wei","family":"Chen","sequence":"additional","affiliation":[{"name":"University of Namur, URBC \u2013 Unit of Research in Cell Biology  Namur Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabienne","family":"George","sequence":"additional","affiliation":[{"name":"CHU UCL Namur, Mont\u2010Godinne Site  Yvoir Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julien","family":"Lemaire","sequence":"additional","affiliation":[{"name":"CHU UCL Namur, Mont\u2010Godinne Site  Yvoir Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5564-1767","authenticated-orcid":false,"given":"Claude","family":"Bertrand","sequence":"additional","affiliation":[{"name":"CHU UCL Namur, Mont\u2010Godinne Site  Yvoir Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laurence","family":"Faugeras","sequence":"additional","affiliation":[{"name":"CHU UCL Namur, Mont\u2010Godinne Site  Yvoir Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8811-8358","authenticated-orcid":false,"given":"Antoine","family":"Fattaccioli","sequence":"additional","affiliation":[{"name":"University of Namur, URBC \u2013 Unit of Research in Cell Biology  Namur Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0445-8353","authenticated-orcid":false,"given":"Quentin","family":"Gilliaux","sequence":"additional","affiliation":[{"name":"CHU UCL Namur, Mont\u2010Godinne Site  Yvoir Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5928-1108","authenticated-orcid":false,"given":"Lionel","family":"D'Hondt","sequence":"additional","affiliation":[{"name":"CHU UCL Namur, Mont\u2010Godinne Site  Yvoir Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9169-1294","authenticated-orcid":false,"given":"Carine","family":"Michiels","sequence":"additional","affiliation":[{"name":"University of Namur, URBC \u2013 Unit of Research in Cell Biology  Namur Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2406-2519","authenticated-orcid":false,"given":"Henri\u2010Fran\u00e7ois","family":"Renard","sequence":"additional","affiliation":[{"name":"University of Namur, URBC \u2013 Unit of Research in Cell Biology  Namur Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3062-3036","authenticated-orcid":false,"given":"Natacha","family":"Zanin","sequence":"additional","affiliation":[{"name":"University of Namur, URBC \u2013 Unit of Research in Cell Biology  Namur Belgium"},{"name":"NARILIS \u2013 NAmur Research Institute for LIfe Sciences  Namur Belgium"},{"name":"GSK  Rixensart Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2023,12,6]]},"reference":[{"key":"e_1_2_13_2_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13058\u2010021\u201001461\u20104"},{"key":"e_1_2_13_3_1","doi-asserted-by":"publisher","DOI":"10.1186\/s40425\u2010019\u20100575\u20103"},{"key":"e_1_2_13_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd.2016.184"},{"key":"e_1_2_13_5_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12943\u2010021\u201001426\u20103"},{"key":"e_1_2_13_6_1","doi-asserted-by":"publisher","DOI":"10.1053\/j.gastro.2011.07.050"},{"key":"e_1_2_13_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature07935"},{"key":"e_1_2_13_8_1","doi-asserted-by":"publisher","DOI":"10.1158\/1078\u20100432.CCR\u201015\u20100124"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.943","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,20]],"date-time":"2024-01-20T04:34:30Z","timestamp":1705725270000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.943"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,12]]},"references-count":7,"journal-issue":{"issue":"12","published-print":{"date-parts":[[2023,12]]}},"alternative-id":["10.1002\/cpz1.943"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.943","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"type":"print","value":"2691-1299"},{"type":"electronic","value":"2691-1299"}],"subject":[],"published":{"date-parts":[[2023,12]]},"article-number":"e943"}}