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LIVER Publication-derived

Novel 3D Approach to Model Non-Alcoholic Fatty Liver Disease using Human Pluripotent Stem Cells

Source Morell et al., 2024 · Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge · 10.7554/elife.95042.1

👤 Carola Maria Morell, Samantha Grace Tilson, Rute Alexandra Tomaz, Arash Shahsavari, Andi Munteanu, Giovanni Canu, Brandon Tyler Wesley, Marion Perrin, Imbisaat Geti, Subhankar Mukhopadhyay, Francesca Mazzacuva, Paul Gissen, Jose Garcia-Bernardo, Martin Bachman, Casey Allison Rimland, Fotios Sampaziotis, Irina Mohorianu, Ludovic Vallier ⏱ 51 days 📋 12 phases 🧫 Human iPSC

Abstract

A multicellular 3D platform using human induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) co-cultured with non-parenchymal cells (cholangiocytes, hepatic stellate cells, and macrophages) in a collagen-based RAFT system. The platform models non-alcoholic fatty liver disease (NAFLD) pathology, including steatosis, lipotoxicity, and multicellular inflammatory and fibrotic responses, using fatty acid treatments to recapitulate chronic hepatic injury.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

46 steps across 12 phases

hiPSC Culture and Maintenance Day 0
  1. 1 Prepare hiPSC culture plates
Hepatocyte-Like Cell (HLC) Differentiation Day 0–23
  1. 1 Differentiate hiPSCs to HLCs
Non-Parenchymal Cell (NPC) Preparation Day 0–7 (M0); ongoing (COs, LX2)
  1. 1 Generate macrophages (M0) from hiPSCs
  2. 2 Obtain cholangiocyte organoids (COs) from primary tissue
  3. 3 Maintain LX2 hepatic stellate cell line
3D RAFT Culture Assembly and Mono-Culture Day 23 (HLC seeding)
  1. 1 Dissociate and prepare HLCs for RAFT seeding
  2. 2 Seed HLCs into RAFT wells
  3. 3 Culture HLCs in 3D for short- or long-term studies
NPC Preparation and Seeding into RAFT Concurrent with HLC seeding
  1. 1 Prepare cholangiocyte organoids for RAFT
  2. 2 Prepare LX2 stellate cells for RAFT
  3. 3 Prepare macrophages (M0) for RAFT
  4. 4 Seed NPCs into RAFT for monocultures
3D Co-Culture Assembly Day 23 (HLC seeding) + same day
  1. 1 Prepare co-culture medium
  2. 2 Seed simple co-cultures (2 cell types)
  3. 3 Seed complex co-cultures (4 cell types)
  4. 4 Maintain 3D co-cultures over time
Fatty Acid Treatment (NAFLD Induction) Day 24–Day 51 (24 hours to 4 weeks post-seeding)
  1. 1 Prepare fatty acid treatments
  2. 2 Apply fatty acid treatment for short-term (24 hours)
  3. 3 Apply fatty acid treatment for intermediate duration (1 week)
  4. 4 Apply fatty acid treatment for chronic duration (4 weeks)
Sample Collection and Preparation for RNA-Seq At 24h and 1-week timepoints
  1. 1 Collect RAFT samples for RNA extraction
  2. 2 Extract total RNA using RNeasy Micro Kit
Bulk RNA-Sequencing After RNA extraction
  1. 1 Perform RNA library preparation
  2. 2 Perform sequencing on Illumina HiSeq v4
  3. 3 Perform quality assessment on raw sequencing data
Single-Cell RNA-Sequencing (scRNA-Seq) At 1-week co-culture timepoint
  1. 1 Prepare RAFT samples for scRNA-seq
  2. 2 Digest collagen matrix
  3. 3 Wash and dissociate cells
  4. 4 Pool technical replicates
  5. 5 Generate single-cell emulsions
  6. 6 Barcode and reverse-transcribe transcripts
  7. 7 Prepare scRNA-seq libraries (V2 chemistry)
scRNA-Seq Data Analysis After sequencing
  1. 1 Perform quality assessment of raw sequencing data
  2. 2 Align reads to human reference genome
  3. 3 Filter cells by quality metrics
  4. 4 Normalize, cluster, and identify marker genes
Functional Assays and Analyses Throughout culture (as specified)
  1. 1 Perform Bodipy493/503 staining for lipid quantification
  2. 2 Assess CYP3A4 activity
  3. 3 Quantify albumin and AFP secretion
  4. 4 Measure viability using PrestoBlue assay
  5. 5 Detect bile acid production
  6. 6 Assess pro-inflammatory and pro-fibrotic cytokine secretion
  7. 7 Perform lipidomic analysis
  8. 8 Perform immunofluorescence staining
  9. 9 Perform quantitative real-time PCR (qPCR)
  10. 10 Perform flow cytometry analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Morell et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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