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LIVER Publication-derived

Liver Organoids for Drug Screening with Vasculature

Full SOP with Academic or a plan

Source Tsang et al., 2021 · MOE Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong · 10.1101/2021.06.29.450347

Authors: Hoi Ying Tsang, Paulisally Hau Yi Lo, Kenneth Ka Ho Lee Duration: 21 days 8 phases Cell source: Human iPSC

Abstract

This protocol describes the differentiation of human induced pluripotent stem cells (hiPSC) into hepatoblasts, mesenchymal stem cells, hepatic stellate cells, and endothelial cells, which self-organize into vascularized liver organoids exhibiting hepatic functions and architecture. The organoids can be induced with thioacetamide or free fatty acids to model liver fibrosis and non-alcoholic fatty liver disease (NAFLD), enabling high-throughput drug screening.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

46 steps across 8 phases

Phase 1 · hiPSC culture and hepatic cell differentiation Days 1-21
  1. a hiPSC culture
  2. b Differentiate hiPSCs to hepatoblasts (HBs)
  3. c Differentiate hiPSCs to mesenchymal stem cells (MSCs)
  4. d Differentiate hiPSCs to hepatic stellate cells (HSCs)
  5. e Differentiate hiPSCs to endothelial cells (ECs)
Phase 2 · Liver organoid differentiation and formation Days 1-14
  1. a Trypsinize and count differentiated cells
  2. b Prepare organoid culture medium
  3. c Seed cells into ultra-low attachment 96-well plates
  4. d Add Y-27632 supplement (days 1-2)
  5. e Add FGF2 supplement (days 1-4)
  6. f Medium replacement (3 times per week)
  7. g Harvest organoids at day 14
Phase 3 · Thioacetamide (TAA) treatment for fibrosis induction Days 1-7 (treatment phase)
  1. a Prepare TAA solutions
  2. b Expose organoids to TAA
  3. c Collect media and organoids
  4. d Assess cell viability
Phase 4 · Free fatty acid (FFA) treatment for steatosis induction Days 1-7 (treatment phase)
  1. a Prepare FFA-BSA conjugates
  2. b Expose organoids to FFA
  3. c Collect media and organoids
  4. d Assess cell viability
Phase 5 · Liver function assays Day 14 (or post-treatment)
  1. a Albumin secretion ELISA
  2. b Indocyanine green (ICG) uptake and excretion
Phase 6 · Molecular analysis - Real-time quantitative PCR (qPCR) Day 14 (or post-treatment)
  1. a RNA extraction
  2. b RNA quantification and purity assessment
  3. c Reverse transcription
  4. d Real-time qPCR
Phase 7 · Immunological analysis - Whole-mount immunofluorescent staining Day 14 (or post-treatment)
  1. a Wash organoids
  2. b Fixation
  3. c Storage
  4. d Permeabilization
  5. e Blocking
  6. f Primary antibody incubation
  7. g Primary antibody wash
  8. h Secondary antibody and DAPI incubation
  9. i Secondary antibody wash
  10. j Clearing solution treatment
  11. k Confocal imaging
Phase 8 · Histological analysis Day 14 (or post-treatment)
  1. a Fixation
  2. b Embedding in agarose-gelatin gel
  3. c Secondary fixation
  4. d Dehydration
  5. e Paraffin embedding
  6. f Sectioning
  7. g Hematoxylin-Eosin (H&E) staining
  8. h Periodic acid-Schiff (PAS) staining
  9. i Pico-sirius red (PS Red) staining

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Attribution

This SOP was written by Organthis based on the published method in Tsang et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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