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LIVER Publication-derived

Recapitulation of hepatitis B virus host interactions in liver organoids from human induced pluripotent stem cells

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

👤 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 ⏱ 42 days 📋 10 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Nie et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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