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LIVER Publication-derived

In situ differentiation and generation of functional liver organoids from human iPSCs in a 3D perfusable chip system

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

👤 Yaqing Wang, Hui Wang, Pengwei Deng, Wenwen Chen, Yaqiong Guo, Tingting Tao, Jianhua Qin ⏱ 48 days 📋 14 phases 🧫 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

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

Full SOP

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Attribution

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

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