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LIVER Publication-derived

Hepatitis B Liver Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Nie et al., 2018 · Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine · 10.1016/j.ebiom.2018.08.014

Authors: Yun-Zhong Nie, Yun-Wen Zheng, Kei Miyakawa, Soichiro Murata, Ran-Ran Zhang, Keisuke Sekine, Yasuharu Ueno, Takanori Takebe, Takaji Wakita, Akihide Ryo, Hideki Taniguchi Duration: 42 days 10 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of functional liver organoids from human induced pluripotent stem cells (hiPSC) by co-culturing hiPSC-derived endoderm with human umbilical vein endothelial cells (HUVECs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) in a 3D microwell system. The resulting organoids exhibit strong hepatic functions and susceptibility to hepatitis B virus (HBV) infection, providing a patient-specific model to study HBV pathogenesis and virus-host interactions.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

35 steps across 10 phases

Phase 1 · Cell Culture Maintenance Prior to differentiation
  1. a Maintain undifferentiated iPSCs
  2. b Prepare HUVECs
  3. c Prepare bone marrow-derived mesenchymal stem cells (BM-MSCs)
Phase 2 · Endoderm Differentiation Days 1-7 (estimated)
  1. a Differentiate hiPSC into endoderm
Phase 3 · Liver Organoid Generation Days 8-22
  1. a Prepare cell suspension for co-culture
  2. b Seed cells into 3D microwell plate
  3. c Culture organoids
  4. d Harvest organoids after 15 days
Phase 4 · HBV Infection Day 22 onwards
  1. a Prepare HBV inoculum
  2. b Infect organoids with HBV
  3. c Culture infected organoids
  4. d Harvest infected organoids
Phase 5 · Viral Quantification and Analysis Days 32-42 (post-infection day 10-20)
  1. a Extract and quantify intracellular viral DNA (vDNA) and covalently closed circular DNA (cccDNA)
  2. b Extract and quantify pregenomic RNA (pgRNA)
  3. c Extract and quantify supernatant viral DNA
Phase 6 · Hepatic Function Assessment Days 1-22 and post-infection
  1. a Measure albumin (ALB) secretion
  2. b Assay urea production
  3. c Assess CYP3A activity
  4. d Measure indocyanine green (ICG) uptake and release
  5. e Measure liver injury markers (ALT and LDH)
Phase 7 · Gene Expression Analysis Throughout culture and infection
  1. a Isolate RNA and perform reverse transcription
  2. b Perform quantitative real-time PCR (Q-PCR)
Phase 8 · Immunofluorescence Analysis Post-infection analysis (day 10-20 post-infection)
  1. a Prepare tissue sections
  2. b Fix and prepare sections for staining
  3. c Apply primary antibodies
  4. d Apply secondary antibody and mount
  5. e Acquire microscopy images
Phase 9 · Transmission Electron Microscopy (TEM) Analysis Post-infection or at end of culture period
  1. a Fix samples for electron microscopy
  2. b Post-fix and dehydrate
  3. c Embed in resin
  4. d Prepare ultrathin sections
  5. e Stain and visualize sections
Phase 10 · Viral Inhibition and Immune Response Studies (Optional) During HBV infection (concurrent with Phase 4)
  1. a HBV entry inhibition
  2. b Reverse transcription inhibition
  3. c Interferon treatment

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Attribution

This SOP was written by Organthis based on the published method in Nie et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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