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LIVER Publication-derived

Liver Organ-on-Chip Models for Drug Screening

Full SOP with Academic or a plan

Source Wang et al., 2018 · Dalian Institute of Chemical Physics, Chinese Academy of Sciences · 10.1039/c8lc00869h

Authors: Yaqing Wang, Hui Wang, Pengwei Deng, Wenwen Chen, Yaqiong Guo, Tingting Tao, Jianhua Qin Duration: 48 days 14 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of functional liver organoids from human induced pluripotent stem cells (hiPSCs) using a 3D perfusable micropillar chip system. The method combines stem cell biology with microengineering to enable in situ hepatic differentiation, producing organoids with hepatocytes and cholangiocytes that exhibit improved gene expression, enhanced liver-specific functions including albumin and urea production, and dose-dependent hepatotoxic response to acetaminophen.

Cell source
Human iPSC
Application
Disease modeling, drug screening, and developmental study

Protocol overview

56 steps across 14 phases

Phase 1 · Device Fabrication and Preparation Prior to cell seeding
  1. a Fabricate PDMS device by soft lithography
  2. b Cure PDMS pre-polymer
  3. c Adhere top layer to bottom layer
  4. d Sterilize chip devices
  5. e Treat chip channels with PF-127 solution
Phase 2 · hiPSC Culture and EB Formation Days -5 to 0
  1. a Maintain hiPSCs in standard culture
  2. b Passage hiPSCs
  3. c Prepare dissociated hiPSCs for EB formation
  4. d Seed cells on micropillar chip for EB formation
Phase 3 · Stage 1: Endoderm Induction (Days 0-5) Days 0-5
  1. a Initiate differentiation protocol on Day 0
  2. b Establish continuous perfusion
  3. c Maintain endoderm differentiation
Phase 4 · Stage 2: Hepatic Progenitor Differentiation and Expansion (Days 5-10) Days 5-10
  1. a Replace medium with growth factor cocktail
  2. b Continue hepatic progenitor culture
Phase 5 · Stage 3: Hepatocyte Maturation (Days 10-15) Days 10-15
  1. a Switch to hepatocyte maturation medium
  2. b Culture organoids with maturation factors
Phase 6 · Stage 4: Long-term Culture and Functional Maturation (Days 15 onwards) Days 15-35
  1. a Transition to long-term maintenance medium
  2. b Maintain long-term culture
Phase 7 · Characterization: Immunohistochemistry and Gene Expression Analysis Days 5, 10, 15, 20, 30
  1. a Harvest organoids for analysis
  2. b Fix organoids for cryosectioning
  3. c Wash and dehydrate organoids
  4. d Embed and freeze organoids
  5. e Prepare cryosections
Phase 8 · Immunofluorescence Staining Protocol Same day as cryosectioning onwards
  1. a Remove OCT and permeabilize sections
  2. b Block non-specific antibody binding
  3. c Incubate with primary antibodies
  4. d Wash and apply secondary antibodies
  5. e Stain cell nuclei
  6. f Acquire and process images
Phase 9 · Real-time Quantitative PCR for Gene Expression Coincident with immunohistochemistry days
  1. a Harvest organoids for RNA extraction
  2. b Extract mRNA using Trizol
  3. c Adjust RNA concentration and quality check
  4. d Synthesize cDNA
  5. e Perform real-time quantitative PCR
  6. f Analyze and quantify gene expression
Phase 10 · Functional Characterization: Albumin and Urea Production Days 15-35
  1. a Collect media samples for albumin measurement
  2. b Measure albumin secretion by ELISA
  3. c Measure urea synthesis
Phase 11 · Functional Characterization: Glycogen and Lipid Storage Day 30
  1. a Prepare cryosections for PAS staining
  2. b Perform PAS staining for glycogen detection
  3. c Perform Oil Red O staining for lipid detection
  4. d Acquire images of stained sections
Phase 12 · Functional Characterization: Cytochrome P450 Enzyme Activity Day 25 onwards (48 hours post-stimulation)
  1. a Stimulate organoids with CYP inducers on Day 25
  2. b Culture organoids with inducers for 48 hours
  3. c Harvest organoids and extract mRNA
  4. d Synthesize cDNA and perform qPCR
  5. e Analyze CYP induction rates
Phase 13 · Drug Toxicity Assessment: Acetaminophen (APAP) Treatment Day 25 onwards (24-48 hours post-exposure)
  1. a Prepare organoids for drug treatment on Day 25
  2. b Expose organoids to APAP at multiple concentrations
  3. c Collect media and assess cell viability
  4. d Measure absorbance at 450 nm
  5. e Analyze dose- and time-dependent toxicity
Phase 14 · Static Culture Control (Parallel to Perfused Culture) Days 0-35 (parallel to perfused culture)
  1. a Set up static culture chip device
  2. b Seed cells and form EBs on static chip
  3. c Differentiate static-cultured organoids with daily media changes
  4. d Characterize static-cultured organoids in parallel

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Attribution

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

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