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LIVER Publication-derived

Engineered Hydrogels for Organoid Models of Human Nonalcoholic Fatty Liver Disease

Source Liu et al., 2025 · Stanford University · 10.1002/advs.202417332

👤 Yueming Liu, Aidan E. Gilchrist, Patrik K. Johansson, Yuan Guan, Jaydon D. Deras, Yu-Chung Liu, Sofia Ceva, Michelle S. Huang, Renato S. Navarro, Annika Enejder, Gary Peltz, Sarah C. Heilshorn ⏱ 19 days 📋 8 phases 🧫 Human iPSC-derived hepatic organoids

Abstract

This protocol describes the development and culture of human iPSC-derived hepatic organoids (HOs) within chemically defined, engineered HELP (hyaluronic acid and elastin-like protein) hydrogels with tunable stiffness to model nonalcoholic fatty liver disease (NAFLD). The protocol includes hydrogel preparation with dynamic stiffening capability via small molecule competitors, hepatic organoid differentiation, and induction of lipid droplet accumulation using oleic acid, enabling mechanistic studies of matrix stiffness effects on hepatocyte lipid metabolism and fibrosis.

Cell source
Human iPSC-derived hepatic organoids
Application
Disease modeling

Protocol overview

74 steps across 8 phases

HA Synthesis and Modification Day 0
  1. 1 Prepare MES buffer
  2. 2 Propargylamine addition
  3. 3 EDC and NHS addition
  4. 4 Overnight reaction
  5. 5 Dialysis
  6. 6 Lyophilization
  7. 7 Copper click reaction setup
  8. 8 Nitrogen degassing
  9. 9 Catalyst preparation
  10. 10 Add catalysts to HA solution
  11. 11 Azido-benzaldehyde coupling
  12. 12 Click reaction
  13. 13 Copper chelation
  14. 14 Post-reaction workup
ELP Expression and Modification Day 0–7
  1. 1 Bacterial transformation
  2. 2 Bacterial culture
  3. 3 ELP induction
  4. 4 Expression period
  5. 5 Cell harvest
  6. 6 ELP extraction via freeze-thaw
  7. 7 Final dialysis
  8. 8 Lyophilization and storage
  9. 9 ELP hydrazine modification setup
  10. 10 Tri-Boc hydrazinoacetic acid activation
  11. 11 Coupling reaction
  12. 12 Precipitation
  13. 13 Drying and Boc removal
  14. 14 Final precipitation and purification
Hydrogel Preparation and Rheological Characterization Day 0–16
  1. 1 Stock solution preparation
  2. 2 Competitor preparation (for HI+ gels)
  3. 3 Laminin addition
  4. 4 Gelation for LO formulation
  5. 5 Gelation for MED formulation
  6. 6 Gelation for HI formulation
  7. 7 Gelation for HI+ formulation
  8. 8 Gel incubation protocol
  9. 9 Rheological characterization setup
  10. 10 Gelation time measurement
  11. 11 Modulus recovery during competitor release
Hepatic Organoid Generation from iPSCs Day 0–7 (initial Matrigel culture)
  1. 1 iPSC-derived hepatoblast preparation
  2. 2 Growth medium preparation
  3. 3 Initial Matrigel culture
  4. 4 Initial growth medium addition
  5. 5 Y-27632 supplementation
  6. 6 Media changes
  7. 7 Spheroid passaging (weekly)
  8. 8 Cell reseeding in Matrigel
Hepatic Organoid Culture in HELP Hydrogels Day 0–16
  1. 1 Organoid release from Matrigel
  2. 2 Cell suspension in ELP-laminin
  3. 3 Hydrogel formation in molds
  4. 4 Gelation protocol
  5. 5 Media addition
  6. 6 Growth phase media changes
  7. 7 Differentiation medium preparation
  8. 8 Differentiation phase media switch
  9. 9 Continued differentiation
Oleic Acid Treatment and Lipid Accumulation Day 16–19
  1. 1 Oleic acid solution preparation
  2. 2 OA-containing medium application
  3. 3 OA incubation period
  4. 4 Control conditions
Mechanosignaling Inhibitor Treatment (Optional) Day 16–19
  1. 1 Y-27632 preparation
  2. 2 Combined OA and Y-27632 treatment
  3. 3 Incubation period
Organoid Harvest and Downstream Analysis Day 19
  1. 1 Hydrogel enzymatic digestion
  2. 2 Cell collection and centrifugation
  3. 3 BODIPY lipid staining
  4. 4 Immunofluorescence staining
  5. 5 RNA extraction for qRT-PCR
  6. 6 cDNA synthesis
  7. 7 qRT-PCR setup and analysis
  8. 8 Triglyceride quantification
  9. 9 Albumin and urea production assays
  10. 10 Cytotoxicity assessment (LDH assay)
  11. 11 CARS microscopy

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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