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LIVER Publication-derived

Generation and Characterization of hPSC-derived Liver Organoids (hPLOs) for Dengue Virus Infection and Antiviral Screening

Source Li et al., 2025 · State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, China · 10.1038/s41467-025-63323-3

👤 Meng-Qi Li, Yan-Peng Xu, Kai Li, Chao Zhou, Xiao-Xuan Fan, Hui Wang, Pan-Deng Shi, Rui-Ting Li, Zu-Xin Wang, Tian-Shu Cao, Qi Chen, Yu-Jun Cui, Yong-Qiang Deng, Xiao-Yan Wu, Hui Zhao, Cheng-Feng Qin ⏱ 50 days 📋 8 phases 🧫 Human iPSC and Human ESC (H1, H9)

Abstract

This protocol describes the generation of functional human pluripotent stem cell-derived liver organoids (hPLOs) containing multiple liver cell types, their characterization, and their application as a platform for modeling dengue virus infection and screening antiviral compounds. hPLOs support productive DENV-2 infection with severe morphological and cellular changes, mimicking clinical dengue pathology.

Cell source
Human iPSC and Human ESC (H1, H9)
Application
Disease modeling and antiviral drug screening

Protocol overview

36 steps across 8 phases

Monolayer Differentiation to Definitive Endoderm (DE) Day 0 to Day 5
  1. 1 Prepare hPSC cultures in E8 medium
  2. 2 Plate hPSC aggregates and initiate DE differentiation
  3. 3 Treat cells with DE medium
Monolayer Differentiation to Hepatic Specification (HS) Day 5 to Day 10
  1. 1 Switch to HS medium
Three-Dimensional (3D) Organoid Formation—Early Stage Day 10 to Day 20–30
  1. 1 Dissociate and prepare cells for 3D embedding
  2. 2 Embed HS cells in Matrigel domes
  3. 3 Culture early hPLOs in basal differentiation medium (BDM)
  4. 4 Passage early hPLOs
  5. 5 Cryopreserve early hPLO fragments
Maturation of hPLOs—Early to Mature Transition Day 20–30 to Day 50
  1. 1 Pretreat early hPLOs with maturation-promoting medium
  2. 2 Dissociate and re-aggregate for mature organoid formation
  3. 3 Culture in mature hepatocyte medium (MHM) with initial supplements
  4. 4 Switch to final mature hepatocyte medium with metabolic factors
Functional Characterization of hPLOs Day 25 to Day 50
  1. 1 Perform whole-mount immunofluorescence staining
  2. 2 Assess glycogen storage by PAS staining
  3. 3 Evaluate indocyanine green (ICG) uptake and release
  4. 4 Assess bile canalicular transport by CDFDA staining
  5. 5 Evaluate MDR1 transporter activity by Rhodamine 123 assay
  6. 6 Measure CYP3A4 and CYP2C9 activity
  7. 7 Quantify secreted proteins and metabolites
  8. 8 Assess cell viability and cytotoxicity by LDH assay
DENV-2 Infection of hPLOs Variable (Day 25 or Day 50 hPLOs used)
  1. 1 Prepare DENV-2 virus inoculum
  2. 2 Prepare hPLO fragments for infection
  3. 3 Inoculate hPLO fragments with DENV-2
  4. 4 Remove inoculum and wash
  5. 5 Re-embed infected fragments in Matrigel
  6. 6 Culture infected organoids
  7. 7 Prepare MOCK (control) group
Antiviral Drug Screening in DENV-2 Infected hPLOs Days 0–5 (infection + treatment)
  1. 1 Prepare drug-treated infected organoid cultures
  2. 2 Monitor organoid morphology and health
  3. 3 Assess functional rescue by PAS staining
  4. 4 Measure metabolic enzyme activity
  5. 5 Quantify albumin and urea secretion
Viral RNA Quantification and Viral Titer Determination Variable (days 1–8 post infection)
  1. 1 Extract viral RNA from culture supernatant
  2. 2 Perform real-time quantitative RT-qPCR
  3. 3 Determine viral titers by plaque assay

Full SOP

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Attribution

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

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