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LIVER Publication-derived

Novel human hepatic organoid model for drug-induced liver fibrosis testing in vitro

Source Leite et al., 2016 · Liver Cell Biology Laboratory, Vrije Universiteit Brussel (VUB), Belgium · 10.1016/j.biomaterials.2015.11.026

👤 Sofia B. Leite, Tiffany Roosens, Adil El Taghdouini, Inge Mannaerts, Ayla J. Smout, Mustapha Najimi, Etienne Sokal, Fozia Noor, Christophe Chesne, Leo A. van Grunsven ⏱ 21 days 📋 5 phases 🧫 Differentiated HepaRG cells (Human hepatocyte-like) and Primary Human Hepatic Stellate Cells (HSCs)

Abstract

This protocol describes the generation of three-dimensional human hepatic organoids by co-culturing differentiated HepaRG cells and primary human hepatic stellate cells in non-attachment 96-well plates with orbital stirring. The resulting scaffold-free organoids (approximately 200 μm diameter) maintain hepatocyte metabolic competence and HSC quiescence for 21 days while enabling detection of drug-induced fibrosis through HSC activation markers, collagen deposition, and hepatocyte-dependent injury responses.

Cell source
Differentiated HepaRG cells (Human hepatocyte-like) and Primary Human Hepatic Stellate Cells (HSCs)
Application
Disease modeling; Drug-induced liver fibrosis testing; Drug screening for hepatotoxicity

Protocol overview

30 steps across 5 phases

Cell preparation and seeding Day 0
  1. 1 Obtain and prepare hepatic cells
  2. 2 Thaw and trypsinize cells
  3. 3 Prepare 3D culture plates
  4. 4 Seed cells into 96-well plates
Spheroid formation and culture maintenance (Days 0-21) Days 0-21
  1. 1 Begin orbital stirring
  2. 2 Minimize evaporation
  3. 3 Culture with initial medium (Days 0-1)
  4. 4 Culture with maintenance medium (Days 1-21)
  5. 5 Perform medium refreshes
  6. 6 Monitor spheroid size
Drug exposure and fibrosis induction (Day 7-21) Days 7-21 (exposure timing varies by compound and assay design)
  1. 1 Prepare for compound exposure (single dose protocol)
  2. 2 Single compound exposure (24 hours)
  3. 3 Prepare for repeated exposure protocol
  4. 4 Repeated compound exposure protocol
  5. 5 Recovery assay (optional)
  6. 6 Inflammatory co-stimulation (optional enhancement)
  7. 7 HDAC inhibitor co-treatment (optional)
Sampling and analysis Days 7, 14, 17, 21
  1. 1 Collect supernatant for secreted protein analysis
  2. 2 Viability assessment (ATP assay)
  3. 3 Gene expression analysis (qPCR)
  4. 4 CYP induction assay
  5. 5 Albumin secretion measurement
  6. 6 Mitochondrial function analysis (Seahorse)
Histological and immunological analysis Days 7, 17, 21
  1. 1 Spheroid fixation and embedding
  2. 2 Immunofluorescence: hepatocyte and HSC markers
  3. 3 Immunofluorescence: HSC activation markers
  4. 4 Collagen immunostaining
  5. 5 Sirius red staining for cross-linked collagen
  6. 6 Caspase-3 immunofluorescence (apoptosis marker)
  7. 7 CMFDA bile accumulation assay (optional)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Leite et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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