Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells

Q2 Immunology and Microbiology

Current Protocols in Immunology Pub Date : 2020-09-17 DOI:10.1002/cpim.106

Cayetano Pleguezuelos-Manzano, Jens Puschhof, Stieneke van den Brink, Veerle Geurts, Joep Beumer, Hans Clevers

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引用次数: 60

Abstract

Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors.

Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies

Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids

Alternate Protocol: Differentiation of human intestinal organoids

Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids

Basic Protocol 4: Immunofluorescence staining of human intestinal organoids

Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures

Support Protocol 1: Production of Wnt3A conditioned medium

Support Protocol 2: Production of Rspo1 conditioned medium

Support Protocol 3: Extraction of RNA from intestinal organoid cultures

Abstract Image

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成体干细胞人肠道类器官的建立与培养

来源于成体干细胞的人类肠道类器官是人类肠道上皮的微型离体版本。肠道类器官是研究肠道生理学和许多疾病状况的有用工具。与长生不老的细胞系相比，这些类器官具有许多优势，但使用它们需要专门的技术。本文中描述的方案为建立和维护来自小肠和结肠活检的人类肠道类器官提供了基本指南。此外，本文还概述了几种人类肠道类器官的下游应用。©2020作者。基本方案1:从新鲜活检组织中建立人小肠和结肠类器官培养基础方案2:人肠道类器官的机械分裂、传代和扩增备用方案:人肠道类器官的分化基本方案3:人肠道类器官的冷冻保存和解冻基本方案4:人肠道类器官的免疫荧光染色基本方案5:单细胞克隆肠道类器官培养的生成支持方案1:Wnt3A条件培养基的生产支持方案2:Rspo1条件培养基的生产支持方案3:从肠道类器官培养物中提取RNA

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Current Protocols in Immunology Immunology and Microbiology-Immunology

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