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OTHER Publication-derived

Organoid Models and Applications in Biomedical Research: Three-Dimensional Culture Systems for Pluripotent Stem Cell-Derived Organoids

Source Xinaris et al., 2015 · IRCCS – Istituto di Ricerche Farmacologiche 'Mario Negri', Bergamo, Italy · 10.1159/000433566

👤 Christodoulos Xinaris, Valerio Brizi, Giuseppe Remuzzi ⏱ 60 days 📋 6 phases 🧫 Human ESC, Human iPSC, Patient-Derived iPSC

Abstract

This protocol describes the generation of complex three-dimensional organoid structures from pluripotent stem cells (PSCs) or progenitor cells through directed differentiation and self-organization. The methods encompass the creation of intestinal, gastric, liver, kidney, brain, and retinal organoids by combining ECM embedding, growth factor signaling, and reaggregation techniques to model organ development and disease in vitro.

Cell source
Human ESC, Human iPSC, Patient-Derived iPSC
Application
Disease modeling, Drug screening, Developmental study, Regenerative medicine

Protocol overview

30 steps across 6 phases

Intestinal Organoid Generation from hESCs/hiPSCs Day 0–14
  1. 1 Induce definitive endoderm formation
  2. 2 Form 3D hindgut spheroids
  3. 3 Culture in pro-intestinal medium with Matrigel
  4. 4 Assess organoid maturation
Gastric Organoid Generation from hESCs/hiPSCs Day 0–21
  1. 1 Induce definitive endoderm
  2. 2 Form endoderm spheroids
  3. 3 Stimulate foregut specification with retinoic acid
  4. 4 Transfer to Matrigel for 3D culture
  5. 5 Verify gastric cell types
Kidney Organoid Generation (Metanephric Approach) from hESCs/hiPSCs Day 0–21
  1. 1 Differentiate hESCs into primitive streak
  2. 2 Induce intermediate mesoderm (IM) specification
  3. 3 Generate ureteric bud (UB) and metanephric mesenchyme (MM) progenitors
  4. 4 Dissociate and reaggregate UB and MM progenitors
  5. 5 Culture reaggregates in 3D culture system
  6. 6 Assess kidney organoid structures
Brain Organoid Generation from hESCs/hiPSCs (Forebrain/Cortical) Day 0–30
  1. 1 Establish floating aggregates resembling embryoid bodies in serum-free medium (SFEB)
  2. 2 Expose aggregates to Wnt and Nodal antagonists
  3. 3 Differentiate to telencephalic precursors
  4. 4 Promote cortical neuron differentiation
  5. 5 Assess neuronal maturation and cortical layering
Retinal Organoid Generation from hESCs/hiPSCs Day 0–60
  1. 1 Generate floating embryoid body-like aggregates
  2. 2 Form optic vesicle-like hemispherical structures
  3. 3 Promote optic cup invagination
  4. 4 Culture OC-organoids for neural retina stratification
  5. 5 Optional: enhance neural retina yield with retinoic acid antagonist
Liver Organoid Generation (Liver Bud) from hiPSCs Day 0–21
  1. 1 Differentiate hiPSCs to endoderm
  2. 2 Prepare human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs)
  3. 3 Co-culture endodermal cells with HUVECs and MSCs in Matrigel
  4. 4 Culture liver buds to promote vascularization and maturation
  5. 5 Assess hepatic function

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Xinaris et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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