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LIVER Publication-derived

Drug-Induced Liver Fibrosis Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 21 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Leite et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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