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LIVER Publication-derived

TNF-α-Promoted Long-Term Culture and Expansion of Primary Mouse Hepatocytes in 3D Matrigel

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

👤 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 ⏱ 180 days 📋 5 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Peng et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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