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LIVER Publication-derived

Liver Organoids for Regenerative Medicine (2018)

Full SOP with Academic or a plan

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

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: 240 days 7 phases Cell source: 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

Phase 1 · Primary Hepatocyte Isolation Day 0
  1. a Cannulate and perfuse liver
  2. b Collagenase perfusion
  3. c Dissect liver and strain
  4. d Low-speed centrifugation
  5. e Percoll gradient purification
Phase 2 · 3D Matrigel Embedding and Culture Initiation Day 0-2
  1. a Prepare basal culture medium
  2. b Prepare expansion medium
  3. c Embed hepatocytes in Matrigel droplets
  4. d Overlay culture medium
  5. e Incubate at 37°C
Phase 3 · Media Changes and Colony Monitoring Day 2-14
  1. a Perform media changes
  2. b Monitor colony formation
  3. c Assess lipid droplet accumulation
Phase 4 · First Passage and Colony Recovery Day 14-21
  1. a Digest Matrigel with dispase
  2. b Collect and wash organoids
  3. c Incubate with TrypLE Express (Passage 1+)
  4. d Re-embed in fresh Matrigel
  5. e Freeze remaining cells (optional)
Phase 5 · Long-Term Culture Maintenance Day 21-240+
  1. a Continue serial passaging
  2. b Supplement with Noggin (Passage 2+)
  3. c Maintain TNF-α throughout culture
  4. d Monitor for lipid accumulation
  5. e Preserve cultures by freezing
Phase 6 · Maturation and Functional Induction (Optional) Variable (e.g., day 21-25 post-passage)
  1. a Prepare EGF-induction medium
  2. b Prepare Wnt-induction medium (alternative)
  3. c Culture in induction medium
  4. d Perform functional assays (optional)
Phase 7 · Transplantation (Optional) Day 14-105+ (timing depends on desired pre-transplant culture duration)
  1. a Prepare hepatocyte organoids for transplant
  2. b Withdraw NTBC from Fah−/− mice (1 day pre-transplant)
  3. c Intrasplenic transplantation
  4. d Manage NTBC cycles post-transplant
  5. e Harvest liver and assess engraftment

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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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