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LIVER Publication-derived

Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids

Source Hu et al., 2018 · Hubrecht Institute, KNAW, Utrecht, the Netherlands · 10.1016/j.cell.2018.11.013

👤 Huili Hu, Helmuth Gehart, Benedetta Artegiani, Carmen López-Iglesias, Florijn Dekkers, Onur Basak, Johan van Es, Susana M. Chuva de Sousa Lopes, Harry Begthel, Jeroen Korving, Maaike van den Born, Chenhui Zou, Corrine Quirk, Luis Chiriboga, Charles M. Rice, Stephanie Ma, Anne Rios, Peter J. Peters, Ype P. de Jong, Hans Clevers ⏱ 140 days 📋 7 phases 🧫 Mouse primary hepatocytes; Human fetal hepatocytes (11-20 weeks gestation); Human primary hepatocytes (pediatric and adult)

Abstract

This protocol describes the establishment and long-term culture of hepatocyte organoids (Hep-Orgs) from primary mouse and human hepatocytes in 3D Matrigel. The organoids retain key hepatocyte functions including albumin secretion, cytochrome P450 activity, and glycogen accumulation, while exhibiting gene expression profiles characteristic of proliferating hepatocytes post-partial hepatectomy. Human hepatocyte organoids successfully engraft and repopulate damaged mouse liver, demonstrating regenerative capacity.

Cell source
Mouse primary hepatocytes; Human fetal hepatocytes (11-20 weeks gestation); Human primary hepatocytes (pediatric and adult)
Application
Disease modeling, Drug screening, Developmental study, Regenerative medicine

Protocol overview

31 steps across 7 phases

Isolation and Preparation of Primary Hepatocytes Day 0
  1. 1 Hepatocyte Isolation by Two-Step Collagenase Perfusion (Mouse)
  2. 2 Hepatocyte Purification by Centrifugation
  3. 3 Cell Counting and Viability Assessment
  4. 4 Hepatocyte Isolation from Human Fetal Liver
Organoid Culture Initiation Day 0-3
  1. 1 Prepare Hep-Medium (Mouse Protocol)
  2. 2 Prepare Hep-Medium (Human Protocol)
  3. 3 Mix Hepatocytes with Matrigel and Seed on Plates
  4. 4 Add Hep-Medium to Solidified Matrigel
  5. 5 Culture Plates in Incubator
Organoid Growth and Medium Refreshment (Days 3-20) Day 3-20
  1. 1 Monitor Organoid Formation
  2. 2 Refresh Medium Every 2-3 Days
  3. 3 Assess Organoid Growth and Morphology
First Passage and Expansion (Day 14-20) Day 14-20
  1. 1 Prepare for Mechanical Disruption (Mouse)
  2. 2 Mechanically Disrupt Organoids
  3. 3 Transfer Disrupted Organoids to Fresh Matrigel
  4. 4 Add Fresh Hep-Medium and Continue Culture
  5. 5 First Passage for Human Hepatocytes (Fetal)
Long-Term Expansion and Maintenance Day 20 onwards
  1. 1 Continue Regular Passages
  2. 2 Monitor Organoid Health and Morphology
  3. 3 Cryopreservation of Organoids (Optional)
Organoid Maturation and Differentiation (Optional for Transplantation) Day -7 to 0 (before transplantation)
  1. 1 Prepare Differentiation Medium for Human Fetal-Orgs
  2. 2 Culture Organoids in Differentiation Medium
  3. 3 Harvest Organoid Cells for Transplantation
Quality Control - Functional and Molecular Characterization Day 20-140 (checkpoints throughout culture)
  1. 1 Periodic Acid-Schiff (PAS) Staining for Glycogen
  2. 2 LDL Uptake Assay
  3. 3 Albumin Secretion ELISA
  4. 4 Cytochrome P450 Activity Assay (CYP1a2 or CYP3A4)
  5. 5 Immunofluorescence Staining for Hepatocyte Markers
  6. 6 Gene Expression Analysis by qRT-PCR
  7. 7 mRNA Sequencing and Analysis
  8. 8 Single-Cell RNA Sequencing Analysis

Full SOP

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Attribution

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

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