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LIVER Publication-derived

Modeling Fibrosis with MASH Patient Liver-Derived Organoids

Source Liu et al., 2025 · University of Pennsylvania, Perelman School of Medicine · 10.1101/2025.09.18.677209;

👤 Andy Liu, A. Dylan T. Haseman, Khushal Bantu, Jia Nong, Vladimir Muzykantov, Jilian Melamed, Michael Kegel, Jenna Muscat-Rivera, Drew Weissman, David Smith, Mei Zhang, Daniel J. Rader, Tobias D. Raabe ⏱ 30 days 📋 10 phases 🧫 Patient-Derived Organoids (MASH), Primary Human Hepatic Stellate Cells, Human Peripheral Blood Monocytes

Abstract

This protocol describes the generation of a 3D human MASH liver fibrosis model by co-culturing organoids derived from metabolic dysfunction-associated steatohepatitis (MASH) patient livers with primary human hepatic stellate cells (HSCs) and peripheral blood monocytes (PBMCs). The resulting organoids develop fibrotic scar-like 3D structures with senescent biphenotypic cells, proliferating myofibroblasts, and proinflammatory macrophages, closely resembling end-stage human MASH fibrotic scars. The model is compatible with lipid nanoparticle (LNP)-mediated gene silencing for therapeutic intervention.

Cell source
Patient-Derived Organoids (MASH), Primary Human Hepatic Stellate Cells, Human Peripheral Blood Monocytes
Application
Disease modeling of MASH-associated liver fibrosis; therapeutic target testing

Protocol overview

57 steps across 10 phases

MASH Liver Tissue Preparation and Organoid Isolation Day 0–5
  1. 1 Tissue Collection and Storage
  2. 2 Tissue Mincing and Washing
  3. 3 Enzymatic Tissue Digestion
  4. 4 Cell Straining and Collection
  5. 5 Basement Membrane Extract (BME) Embedding
  6. 6 Overlay with Isolation Medium
Organoid Expansion and Passaging Day 5–30
  1. 1 Mechanical Dissociation and Cell Separation
  2. 2 BME Dissolution and Cell Collection
  3. 3 Re-embedding in Fresh BME and Expansion
  4. 4 Overlay with Pre-warmed Expansion Medium
  5. 5 ROCK Inhibitor Supplementation for Frozen Cell Vials
Organoid Differentiation and 2D Monolayer Formation Day 0–12
  1. 1 Prepare BME-Coated 96-Well Plates
  2. 2 Mechanically Disassemble Organoids
  3. 3 BME Dissolution and Cell Recovery
  4. 4 Resuspend in Organoid Differentiation Medium and Plate
  5. 5 Culture in Differentiation Medium
Primary Hepatic Stellate Cell (HSC) Culture and Differentiation into Myofibroblasts (MFBs) Day 0–7
  1. 1 Prepare BME-Coated 24-Well Plates
  2. 2 Thaw and Prepare Primary HSCs
  3. 3 Prepare Fibrosis Medium
  4. 4 Plate HSCs in Fibrosis Medium
  5. 5 Culture HSCs for Differentiation into MFBs
  6. 6 Routine MFB Passaging (Optional)
Human Peripheral Blood Monocyte (MC) Isolation and Storage Day 0 (Collection)–Day 30+ (Use)
  1. 1 MC Isolation via Negative Selection
  2. 2 MC Preparation for Freezing
  3. 3 MC Thawing for Use
Triple Co-Culture Setup for 3D MASH Fibrosis Model Day 0–5 (Initial assembly)–Day 15 (Full culture)
  1. 1 Establish Organoid Cell Monocultures on 96-Well Plates
  2. 2 Prepare MFB and MC Solutions
  3. 3 Add MFBs to Organoid Monolayers
  4. 4 Add MCs to Establish Triple Co-Culture
  5. 5 Culture Triple Co-Cultures in Fibrosis Medium
  6. 6 Optional: Add Free Fatty Acids to Enhance MASH Phenotype
LNP-Mediated Gene Silencing (YAP1 siRNA Knockdown) Day 2–5 (LNP treatment)–Day 7 (Harvest)
  1. 1 Prepare LNPs conjugated to an MFB-specific antibody. Load these LNPs with anti-YAP1 siRNA.
  2. 2 Prepare and Modify MFB-Specific Antibody
  3. 3 Conjugate DBCO-Antibodies to Azide-Functionalized LNPs
  4. 4 Sterilize LNP-Antibody Conjugates
  5. 5 Add LNPs to Triple Co-Cultures
  6. 6 Culture with LNPs and Assess Knockdown
Sample Preparation, Immunofluorescence Staining, and Imaging Day 7–15 (Final culture)–Harvest
  1. 1 Fix Cells with Formalin
  2. 2 Permeabilize Cells with Triton X-100
  3. 3 Block with Bovine Serum Albumin (BSA)
  4. 4 Primary Antibody Staining
  5. 5 Secondary Antibody Staining
  6. 6 Final Wash and Mounting
  7. 7 Image Acquisition on Inverted Fluorescence Microscope
  8. 8 Image Processing and Analysis
Single-Cell RNA Sequencing (scRNA-seq) Sample Preparation Day 7 (Triple co-culture)–Harvest
  1. 1 Incubate Triple Co-Cultures in PBS (−/−)
  2. 2 Enzymatic Dissociation with TrypLE
  3. 3 Cell Recovery and Resuspension
  4. 4 Transfer to Single-Cell Core Facility
  5. 5 10x Genomics Chromium Library Preparation
Single-Cell RNA Sequencing Data Analysis Post-Sequencing (Data-dependent)
  1. 1 Raw Data Processing and Alignment
  2. 2 Data Quality Control and Preprocessing
  3. 3 Integration and Cell Clustering
  4. 4 Cell Type Annotation Using Reference Data
  5. 5 Differential Gene Expression and Pathway Analysis
  6. 6 Ligand-Receptor Pair Analysis
  7. 7 Visualization and Reporting

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Liu et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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