Collection 27 February 2023

Organoid protocols

Organoid culture systems are self-renewing 3D models derived from stem cells (pluripotent or adult-derived) or cancer cells that recapitulate characteristics of their tissues of origin. Here, we provide protocols for growing and studying various types of organoid (brain, skin, kidney, liver, bile duct, prostate, placenta, breast, colon, stomach, pancreas, lung, heart, and blood vessel) generated from both healthy and cancer cells.

Editors

Mel Clyne &

Mel Clyne

Nature Protocols
Andrew Economou

Andrew Economou

Nature Protocols

Protocols

Intestinal organoid cocultures with microbes

This protocol comprises various methods to coculture organoids (particularly human small intestinal and colon organoids) with microbes, including microinjection into the lumen and periphery of 3D organoids and exposure of organoids to microbes in a 2D layer.
- Jens Puschhof
- Cayetano Pleguezuelos-Manzano
- Hans Clevers
Protocol11 Aug 2021 Nature Protocols
Establishment of patient-derived cancer organoids for drug-screening applications

This protocol summarizes the various approaches available to derive organoids from cancer patients and use these for screening of possible treatments. An optimized protocol for using head and neck cancer organoids is also described.
- Else Driehuis
- Kai Kretzschmar
- Hans Clevers
Protocol14 Sep 2020 Nature Protocols
Multiplexed single-cell analysis of organoid signaling networks

This multiplexed mass cytometry protocol uses thiol-reactive organoid barcoding in situ and a cytometry by time of flight signaling analysis pipeline (CyGNAL) to enable 126-plex single-cell analysis of cell type, cell state and post-translational modification signaling networks in organoids.
- Jahangir Sufi
- Xiao Qin
- Christopher J. Tape
Protocol8 Sep 2021 Nature Protocols
Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids

Dekkers et al. provide a toolbox for the long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.
- Johanna F. Dekkers
- Esmée J. van Vliet
- Anne C. Rios
Protocol10 Mar 2021 Nature Protocols
Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds

The authors present a protocol for bioengineering human intestinal mucosal grafts. This includes the isolation, expansion and biobanking of patient-derived intestinal organoids and fibroblasts and the decellularization and recellularization of human intestinal scaffolds.
- Laween Meran
- Lucinda Tullie
- Vivian S. W. Li
Protocol19 Oct 2022 Nature Protocols
Coculture of primary human colon monolayer with human gut bacteria

The protocol describes coculture of a primary human colon monolayer derived from cells growing in colon organoids with aerotolerant bacteria, such as strains of B. thetaiotaomicron genetically engineered to respond to different stimuli in colonic microenviroments.
- Jianbo Zhang
- Victor Hernandez-Gordillo
- Linda G. Griffith
Protocol28 Jun 2021 Nature Protocols
Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

Skin organoids are generated from human pluripotent stem cells in a three-dimensional in vitro culture system. The mature organoids contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells and sensory neurons.
- Jiyoon Lee
- Wouter H. van der Valk
- Karl R. Koehler
Protocol23 Mar 2022 Nature Protocols
3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert

The authors describe how to make both a gut-on-a-chip and a hybrid chip with a Transwell insert, and how to induce 3D morphogenesis of human intestinal epithelium from either Caco-2 cells or organoids using basolateral medium flow in both platforms.
- Woojung Shin
- Hyun Jung Kim
Protocol2 Feb 2022 Nature Protocols
Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development

In this Protocol Extension, Lancaster et al. describe a modified version of their original protocol (published in 2014) that can be used to reliably generate cerebral organoids of a telencephalic identity and maintain long-term viability for later stages of neural development, including axon outgrowth and neuronal maturation.
- Stefano L. Giandomenico
- Magdalena Sutcliffe
- Madeline A. Lancaster
Protocol Extension16 Dec 2020 Nature Protocols
Transplantation of intestinal organoids into a mouse model of colitis

An approach to studying epithelia derived from organoids of either fetal or adult state and from both the small intestine and colon via transplantation into animals in which the colon has been damaged following administration of dextran sulfate sodium.
- Satoshi Watanabe
- Sakurako Kobayashi
- Shiro Yui
Protocol2 Feb 2022 Nature Protocols
Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells

Takebe et al. describe a protocol for the continuous patterning of hepatic, biliary and pancreatic structures from a 3D culture of human pluripotent stem cells.
- Hiroyuki Koike
- Kentaro Iwasawa
- Takanori Takebe
Protocol11 Jan 2021 Nature Protocols
A well plate–based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature

This protocol describes how to integrate organoids into a vascularized organ-on-a-chip biofabricated platform. The platform is assembled in a 96-well format and allows the connection of two tissues through a single endothelialized microchannel blood vessel.
- Benjamin Fook Lun Lai
- Rick Xing Ze Lu
- Milica Radisic
Protocol31 Mar 2021 Nature Protocols
Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation

This protocol describes the long-term culture of liver and pancreas 3D organoids from human and mouse, and differentiation of liver organoids in vitro and in vivo. Methodology for genetic manipulation of these self-renewing organoids is also detailed.
- Laura Broutier
- Amanda Andersson-Rolf
- Meritxell Huch
Protocol25 Aug 2016 Nature Protocols
Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue

Bioengineered biliary tissue suitable for biliary reconstruction is obtained from cholangiocyte organoids derived from human primary extra- or intrahepatic duct cholangiocytes.
- Olivia C. Tysoe
- Alexander W. Justin
- Fotios Sampaziotis
Protocol20 May 2019 Nature Protocols
Generation of kidney organoids from human pluripotent stem cells

This protocol describes stepwise differentiation of human pluripotent stem cells into 3D kidney organoids that contain segmented nephrons connected to collecting ducts, which are surrounded by renal interstitial cells and an endothelial network.
- Minoru Takasato
- Pei X Er
- Melissa H Little
Protocol18 Aug 2016 Nature Protocols
Generation of blood vessel organoids from human pluripotent stem cells

Self-organizing 3D human blood vessel organoids are generated by mesoderm induction of hPSC aggregates and subsequent differentiation into endothelial networks and pericytes in a 3D collagen I–Matrigel matrix.
- Reiner A. Wimmer
- Alexandra Leopoldi
- Josef M. Penninger
Protocol25 Sep 2019 Nature Protocols
Generation of lung organoids from human pluripotent stem cells in vitro

Human pluripotent stem cells are differentiated into ventral–anterior foregut spheroids and then to lung organoids, resembling the bronchi and surrounding mesenchyme of the developing human airway, or bud tip progenitor organoids.
- Alyssa J. Miller
- Briana R. Dye
- Jason R. Spence
Protocol21 Jan 2019 Nature Protocols
Organoid culture systems for prostate epithelial and cancer tissue

This protocol describes a strategy for generating 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FAC-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells.
- Jarno Drost
- Wouter R Karthaus
- Hans Clevers
Protocol21 Jan 2016 Nature Protocols
Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta

This protocol describes how to isolate trophoblast from first-trimester human placentas and grow it long term in a three-dimensional organoid culture system.
- Megan A. Sheridan
- Ridma C. Fernando
- Margherita Y. Turco
Protocol9 Sep 2020 Nature Protocols
Generation of heart-forming organoids from human pluripotent stem cells

Human pluripotent stem cell aggregates are formed, embedded in Matrigel and directed to differentiate to heart-forming organoids by the chemical WNT pathway modulators CHIR99021 and IWP2.
- Lika Drakhlis
- Santoshi Biswanath Devadas
- Robert Zweigerdt
Protocol10 Nov 2021 Nature Protocols

Organoid protocols

Editors

Protocols

Intestinal organoid cocultures with microbes

Establishment of patient-derived cancer organoids for drug-screening applications

Multiplexed single-cell analysis of organoid signaling networks

Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids

Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds

Coculture of primary human colon monolayer with human gut bacteria

Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert

Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development

Transplantation of intestinal organoids into a mouse model of colitis

Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells

A well plate–based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature

Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation

Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue

Generation of kidney organoids from human pluripotent stem cells

Generation of blood vessel organoids from human pluripotent stem cells

Generation of lung organoids from human pluripotent stem cells in vitro

Organoid culture systems for prostate epithelial and cancer tissue

Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta

Generation of heart-forming organoids from human pluripotent stem cells

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Organoid protocols

Editors

Mel Clyne

Andrew Economou

Protocols

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