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LIVER Publication-derived

TNF-α Promotes Long-Term Expansion of Primary Hepatocytes in 3D Culture

Source Peng et al., 2018 · Howard Hughes Medical Institute, Department of Developmental Biology, 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 ⏱ 240 days 📋 7 phases 🧫 Primary mouse hepatocytes (C57BL/6J)

Abstract

This protocol describes the establishment and long-term expansion of primary mouse hepatocytes in 3D Matrigel culture using TNF-α as a key expansion signal. The culture system maintains hepatocyte identity, functionality, and engraftment potential, enabling serial passaging for over 6 months. In vivo transplantation into Fah−/− mice demonstrates robust liver repopulation and regeneration.

Cell source
Primary mouse hepatocytes (C57BL/6J)
Application
Hepatocyte expansion and long-term culture for regenerative medicine and transplantation studies

Protocol overview

32 steps across 7 phases

Primary Hepatocyte Isolation Day 0
  1. 1 Cannulate and perfuse liver
  2. 2 Collagenase perfusion
  3. 3 Dissect liver and strain
  4. 4 Low-speed centrifugation
  5. 5 Percoll gradient purification
3D Matrigel Embedding and Culture Initiation Day 0–2
  1. 1 Prepare basal culture medium
  2. 2 Prepare expansion medium
  3. 3 Embed hepatocytes in Matrigel droplets
  4. 4 Overlay culture medium
  5. 5 Incubate at 37°C
Media Changes and Colony Monitoring Day 2–14
  1. 1 Perform media changes
  2. 2 Monitor colony formation
  3. 3 Assess lipid droplet accumulation
First Passage and Colony Recovery Day 14–21
  1. 1 Digest Matrigel with dispase
  2. 2 Collect and wash organoids
  3. 3 Incubate with TrypLE Express (Passage 1+)
  4. 4 Re-embed in fresh Matrigel
  5. 5 Freeze remaining cells (optional)
Long-Term Culture Maintenance Day 21–240+
  1. 1 Continue serial passaging
  2. 2 Supplement with Noggin (Passage 2+)
  3. 3 Maintain TNF-α throughout culture
  4. 4 Monitor for lipid accumulation
  5. 5 Preserve cultures by freezing
Maturation and Functional Induction (Optional) Variable (e.g., day 21–25 post-passage)
  1. 1 Prepare EGF-induction medium
  2. 2 Prepare Wnt-induction medium (alternative)
  3. 3 Culture in induction medium
  4. 4 Perform functional assays (optional)
Transplantation (Optional) Day 14–105+ (timing depends on desired pre-transplant culture duration)
  1. 1 Prepare hepatocyte organoids for transplant
  2. 2 Withdraw NTBC from Fah−/− mice (1 day pre-transplant)
  3. 3 Intrasplenic transplantation
  4. 4 Manage NTBC cycles post-transplant
  5. 5 Harvest liver and assess engraftment

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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