[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"detail-sidebar-cat-0-en-105":3,"doc-seo-451091-105":59,"doc-detail-451091-en":131},{"code":4,"msg":5,"data":6},0,"success",[7,13,18,23,28,33,38,43,48,51,55],{"id":8,"doc_module":4,"doc_module_name":9,"category_name":10,"show_sort_weight":11,"slug":12},1,"Document","Story & Novel",90,"story-novel",{"id":14,"doc_module":4,"doc_module_name":9,"category_name":15,"show_sort_weight":16,"slug":17},2,"Literature",80,"literature",{"id":19,"doc_module":4,"doc_module_name":9,"category_name":20,"show_sort_weight":21,"slug":22},4,"Exam",70,"exam",{"id":24,"doc_module":4,"doc_module_name":9,"category_name":25,"show_sort_weight":26,"slug":27},5,"Comic",60,"comic",{"id":29,"doc_module":4,"doc_module_name":9,"category_name":30,"show_sort_weight":31,"slug":32},6,"Technology",50,"technology",{"id":34,"doc_module":4,"doc_module_name":9,"category_name":35,"show_sort_weight":36,"slug":37},7,"Healthcare",40,"healthcare",{"id":39,"doc_module":4,"doc_module_name":9,"category_name":40,"show_sort_weight":41,"slug":42},8,"Research & Report",30,"research-report",{"id":44,"doc_module":4,"doc_module_name":9,"category_name":45,"show_sort_weight":46,"slug":47},9,"Religion & Spirituality",20,"religion-spirituality",{"id":46,"doc_module":4,"doc_module_name":9,"category_name":49,"show_sort_weight":46,"slug":50},"World Cup","world-cup",{"id":52,"doc_module":4,"doc_module_name":9,"category_name":53,"show_sort_weight":52,"slug":54},10,"Lifestyle","lifestyle",{"id":56,"doc_module":4,"doc_module_name":9,"category_name":57,"show_sort_weight":24,"slug":58},19,"General","general",{"code":4,"msg":60,"data":61},"ok",{"site_id":62,"language":63,"slug":64,"title":65,"keywords":66,"description":67,"schema_data":68,"social_meta":124,"head_meta":126,"extra_data":128,"updated_unix":130},105,"en","protocol-for-the-preparation-and-analysis-of-patient-derived-organotypic-tumor-spheroids","Protocol for the preparation and analysis of patient-derived organotypic tumor spheroids","","Patient-derived organotypic tumor spheroids (PDOTS) are short-term three-dimensional multicellular cultures generated from freshly explanted patient tumors using microfluidic devices. This protocol outlines PDOTS sample processing and culturing, ex vivo drug-sensitivity testing with a microscopy-based assay, and the assembly of a PDOTS cohort designed to capture overall responses while reflecting patient-to-patient diversity. It emphasizes critical technical steps, highlights common pitfalls, and provides troubleshooting and quality-control guidance. It also clarifies how PDOTS retain tumor-infiltrating immune and stromal cells to support translational tumor-immune interaction studies and immunotherapy testing.",{"@graph":69,"@context":123},[70,84,106],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":35,"@type":76,"position":81},"https://docshare.wps.com/document/healthcare/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/protocol-for-the-preparation-and-analysis-of-patient-derived-organotypic-tumor-spheroids/451091/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/protocol-for-the-preparation-and-analysis-of-patient-derived-organotypic-tumor-spheroids/451091.png","ImageObject",300,407,{"name":92,"@type":93},"Chumphorn","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-11-08","2026-09-30",true,{"@type":102,"interactionType":103,"userInteractionCount":105},"InteractionCounter",{"@type":104},"ViewAction",12,{"@type":107,"mainEntity":108},"FAQPage",[109,115,119],{"name":110,"@type":111,"acceptedAnswer":112},"What are patient-derived organotypic tumor spheroids (PDOTS) and how are they generated?","Question",{"text":113,"@type":114},"PDOTS are short-term, 3D multicellular spheroid cultures prepared from freshly explanted patient tumors using microfluidic devices. They are generated through PDOTS sample processing and culturing steps described in the protocol.","Answer",{"name":116,"@type":111,"acceptedAnswer":117},"How does the protocol test immunotherapy or drug efficacy ex vivo?",{"text":118,"@type":114},"It uses an ex vivo microscopy-based assay to assess drug sensitivity. The protocol also describes assembling a PDOTS cohort that captures overall responses with patient-to-patient diversity.",{"name":120,"@type":111,"acceptedAnswer":121},"What biosafety and ethical requirements must be followed?",{"text":122,"@type":114},"Human tissue use should comply with national and institutional ethical guidelines, including the Declaration of Helsinki and IRB approval with informed consent. Experiments should follow BSL2 biosafety practices, including using a class II biosafety cabinet and appropriate PPE, and preparing solutions and buffers in advance.","https://schema.org",{"og:url":83,"og:type":125,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":127,"canonical":83},"index,follow",{"doc_id":129,"site_id":62},451091,1791351637,{"code":4,"msg":5,"data":132},{"doc_id":129,"user_id":133,"nickname":92,"user_avatar":134,"doc_module":4,"category_id":34,"category_name":35,"doc_title":65,"doc_description":67,"doc_content":135,"file_id":136,"file_url":137,"file_type":138,"file_size":139,"view_count":105,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":140,"language":141,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":142,"faqs":143,"seo_title":144,"seo_description":67,"update_tm":145,"read_time":146,"preview_page_count":4},2336475401981,"https://ap-avatar.wpscdn.com/avatar/22000c94efd8d5204d?x-image-process=image/resize,m_fixed,w_180,h_180&k=1786935347598174694","ll  \nOPEN ACCESS  \nProtocol  \nProtocol for the preparation and analysis of patient-derived organotypic tumor spheroids  \nClaire A. Palin, Russell W. Jenkins, Or-Yam Revach  \n[jenkinr@musc.edu](jenkinr@musc.edu)  \nHighlights  \nSteps for isolating immune-competent PDOTS from freshly explanted human tumors  \nInstructions for testing the efficacy of immunotherapies ex vivo  \nDownstream flow and microscopy-based immunophenotyping  \nGuidance for ex vivo PDOTS cohort assembly of therapies and therapy combinations  \nPalin et al. , STAR Protocols 7, 104286  \nMarch 20, 2026 © 2025 The Authors. Published by Elsevier Inc.  \n[https://doi.org/10.1016/](https://doi.org/10.1016/)[ ](https://doi.org/10.1016/)[j.xpro.2025.104286](j.xpro.2025.104286)  \nll  \nOPEN ACCESS  \nProtocol  \nProtocol for the preparation and analysis of patientderived organotypic tumor spheroids  \nClaire A. Palin, 1 Russell W. Jenkins, 1,2,3,4,6, * and Or-Yam Revach1,2,3,5  \n1Mass General Cancer Center, Krantz Family Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA  \n2Harvard Medical School, Boston, MA 02115, USA  \n3Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA  \n4Present address: Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA  \n5Technical contact  \n6Lead contact  \n*Correspondence: [jenkinr@musc.edu](jenkinr@musc.edu)  \n[https://doi.org/10.1016/j.xpro.2025.104286](https://doi.org/10.1016/j.xpro.2025.104286)  \nSUMMARY  \nPatient-derived organotypic tumor spheroids (PDOTS) are short-term, 3D cultures of multicellular spheroids in microfluidic devices, prepared from freshly explanted patient tumors. In this protocol, we describe the steps of PDOTS sample processing and culturing, drug sensitivity testing ex vivo using a microscopybased assay, and the assembly of a PDOTS cohort that captures the overall response with patient-to-patient diversity. We highlight critical technical steps and call attention to potential pitfalls, offering troubleshooting guidance and  \nimportant quality control details.  \nFor complete details on the use and execution of this protocol, please refer to  \nRevach et al.1  \nBEFORE YOU BEGIN  \nOur step-by-step protocol provides readers with a detailed description of key steps in generating, culturing, treating, and evaluating PDOTS isolated from freshly resected human tumors. Unlike patient-derived organoids (PDOs), PDOTS retain tumor-infiltrating immune and stromal cells, enabling the study of dynamic tumor-immune interactions and performing immunotherapy drug sensitivity testing using clinically relevant biospecimens. With the FDA encouraging the use of new alternative methods (NAMs), like organ-on-a-chip, human ex vivo models, and in silico models to test new drugs and the NIH prioritization of human-focused research,2 this protocol will be of particular benefit for tumor immunologists with an interest in using a clinically relevant, translational model.  \nPrevious studies demonstrated that PDOTS contains all cell types that are present in the original tumor, including immune cells, and that the ex vivo responses to immune checkpoint blockade (ICB) closely align with in vivo outcomes. The method was validated across multiple solid tumor types, including melanoma, colon cancer, pancreatic ductal adenocarcinoma (PDAC), and head and neck squamous cell carcinoma (HNSCC), as well as various treatment conditions and combinations, including ICB monoclonal antibodies, CAR-T cell therapy, small molecules, and viral vector gene therapy.1 ,3–9 Since every tumor has unique properties, different tumors might requireslight method calibration. For example, more fibrotic tumors will require an additional collagenase degradation time (up to 60 min) . Yet, this protocol is highly reproducible with multiple solid tumors.  \nSTAR Protocols 7, 104286, March 20, 2026 © 2025 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND lice","cbCaivXa1wh0ReMI","https://ap.wps.com/l/cbCaivXa1wh0ReMI","pdf",6035170,22,"English","# SUMMARY\n# BEFORE YOU BEGIN\n## Key concept and model rationale\n# INNOVATION\n# Institutional permissions\n# Biosafety considerations\n# Human subjects research","[{\"question\":\"What are patient-derived organotypic tumor spheroids (PDOTS) and how are they generated?\",\"answer\":\"PDOTS are short-term, 3D multicellular spheroid cultures prepared from freshly explanted patient tumors using microfluidic devices. They are generated through PDOTS sample processing and culturing steps described in the protocol.\"},{\"question\":\"How does the protocol test immunotherapy or drug efficacy ex vivo?\",\"answer\":\"It uses an ex vivo microscopy-based assay to assess drug sensitivity. The protocol also describes assembling a PDOTS cohort that captures overall responses with patient-to-patient diversity.\"},{\"question\":\"What biosafety and ethical requirements must be followed?\",\"answer\":\"Human tissue use should comply with national and institutional ethical guidelines, including the Declaration of Helsinki and IRB approval with informed consent. Experiments should follow BSL2 biosafety practices, including using a class II biosafety cabinet and appropriate PPE, and preparing solutions and buffers in advance.\"}]","Protocol for the preparation and analysis of patient-derived organotypic tumor spheroids | PDF",1790734353,55]