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LIVER Publication-derived

Liver Organoids for Regeneration and Long-Term Culture

Full SOP with Academic or a plan

Source Peng et al., 2018 · Stanford University School of Medicine · 10.1016/j.cell.2018.11.012

Authors: Weng Chuan Peng, Catriona Y. Logan, Matt Fish, Teni Anbarchian, Francis Aguisanda, Adrián Álvarez-Varela, Peng Wu, Yinhua Jin, Junjie Zhu, Bin Li, Markus Grompe, Bruce Wang, Roel Nusse Duration: 180 days 5 phases Cell source: Primary mouse hepatocytes

Abstract

This protocol enables long-term 3D culture and serial passaging of primary mouse hepatocytes for >6 months using TNF-α in combination with growth factors and Wnt signaling. The expanded hepatocytes retain hepatocyte identity and function, can be differentiated to enhance functional gene expression, and successfully engraft injured livers of Fah−/− mice with robust repopulation.

Cell source
Primary mouse hepatocytes
Application
Disease modeling and regenerative medicine; long-term hepatocyte expansion

Protocol overview

24 steps across 5 phases

Phase 1 · Primary Hepatocyte Isolation Day 0
  1. a Cannulation and liver perfusion
  2. b Collagenase digestion
  3. c Dissection and straining
  4. d Low-speed centrifugation for hepatocyte enrichment
  5. e Percoll gradient purification
Phase 2 · 3D Culture Initiation and Expansion Days 1-14
  1. a Preparation of basal culture medium
  2. b Preparation of expansion medium
  3. c Matrigel droplet preparation and cell embedding
  4. d Initial culture phase
  5. e Assessment of colony formation
Phase 3 · First Passage and Continued Expansion Days 15-35
  1. a Matrigel digestion with dispase
  2. b Optional TrypLE treatment for cell aggregate dissociation
  3. c Re-embedding in fresh Matrigel
  4. d Continued culture with media changes
  5. e Cryopreservation of cells
Phase 4 · Hepatocyte Maturation/Induction (Optional) Days 21-25 (4 days of induction following ~2 weeks expansion)
  1. a Preparation of EGF-induction medium (periportal gene induction)
  2. b Preparation of Wnt-induction medium (pericentral gene induction)
  3. c Switch to induction medium
  4. d Assessment of functional gene upregulation
Phase 5 · Quality Control and Validation Throughout culture (days 1-180+)
  1. a Morphological assessment
  2. b Immunofluorescence validation of hepatocyte identity
  3. c Lipid droplet quantification
  4. d Gene expression validation by qPCR
  5. e TNF-α dependency confirmation

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Attribution

This SOP was written by Organthis based on the published method in Peng et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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