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LIVER Publication-derived

Combined effect of polystyrene microplastics and bisphenol A on human embryonic stem cell-derived liver organoids: hepatotoxicity and lipid accumulation assessment

Source Cheng et al., 2023 · Shanghai Jiao Tong University School of Medicine · 10.1016/j.scitotenv.2022.158585

👤 Wei Cheng, Yue Zhou, Yichun Xie, Yan Li, Ren Zhou, Hui Wang, Yan Feng, Yan Wang ⏱ 27 days 📋 10 phases 🧫 Human ESC (H1 ES, WA01)

Abstract

This protocol describes the generation of human embryonic stem cell-derived liver organoids and their exposure to polystyrene microplastics (1 μm) and bisphenol A to assess combined hepatotoxicity and lipid metabolism disruption. The protocol employs dynamic exposure in spinner flasks and measures hepatic injury markers, oxidative stress, inflammatory response, and lipid accumulation.

Cell source
Human ESC (H1 ES, WA01)
Application
Disease modeling

Protocol overview

58 steps across 10 phases

hPSC Cultivation and Hepatic Differentiation Day 0-18
  1. 1 Culture human embryonic stem cells (H1 ES)
  2. 2 Dissociate H1 ES cells into single cells
  3. 3 Initiate hepatic differentiation (Days 0-5)
  4. 4 Continue hepatic differentiation with growth factors (Days 5-18)
Liver Organoid Generation in Spinner Flasks Day 18-24
  1. 5 Transfer hepatocytes to spinner flasks
  2. 6 Initiate spinner flask rotation (Days 18-21)
  3. 7 Filter and select cell spheroids by size
  4. 8 Continue culturing selected spheroids (Days 21-24)
Cytotoxicity Determination and Concentration Selection Day 24-27
  1. 9 Prepare organoids for cytotoxicity assay
  2. 10 Expose organoids to varying PS concentrations
  3. 11 Expose organoids to varying BPA concentrations
  4. 12 Collect supernatant and measure LDH release
  5. 13 Normalize LDH data and calculate IC values
  6. 14 Perform calcein/PI staining for visual cytotoxicity assessment
Dynamic Exposure to PS and BPA in Spinner Flasks Day 24-27
  1. 15 Prepare BPA and PS stock solutions
  2. 16 Set up exposure groups with 4×4 concentration combinations
  3. 17 Add PS and BPA to organoid cultures in spinner flasks
  4. 18 Perform dynamic exposure with controlled rotation
  5. 19 Collect supernatant medium samples during exposure
  6. 20 Quantify PS particles in medium by flow cytometry
BPA Bioavailability Analysis Day 24-27
  1. 21 Prepare BPA media samples without serum
  2. 22 Prepare paired BPA and BPA + PS samples
  3. 23 Incubate samples for 72 hours
  4. 24 Collect media and prepare for BPA analysis
  5. 25 Measure BPA concentrations by HPLC-MS/MS
  6. 26 Compare BPA concentrations between paired samples
Organoid Characterization and Microscopy Day 18-27
  1. 27 Prepare frozen sections of organoids
  2. 28 Perform immunofluorescent staining
  3. 29 Photograph immunofluorescent samples
  4. 30 Perform Scanning Electron Microscopy (SEM)
  5. 31 Perform Transmission Electron Microscopy (TEM)
Gene Expression and Molecular Analysis Day 27 (post-exposure)
  1. 32 Extract total RNA from organoids
  2. 33 Perform reverse transcription
  3. 34 Perform quantitative real-time PCR (qRT-PCR)
  4. 35 Calculate relative gene expression
Protein Analysis and Enzyme Activity Day 27 (post-exposure)
  1. 36 Prepare organoid lysates for protein analysis
  2. 37 Perform Western blotting
  3. 38 Measure CYP3A4 enzyme activity
  4. 39 Measure hepatic injury markers (AST, ALT)
  5. 40 Measure oxidative stress markers
  6. 41 Measure ATP content
  7. 42 Measure inflammatory markers (IL-6)
  8. 43 Measure secreted albumin (ALB)
  9. 44 Measure β-glucosidase (GUSβ)
Lipid Accumulation and Metabolism Analysis Day 27 (post-exposure)
  1. 45 Pre-incubate organoids with palmitic acid
  2. 46 Measure total triglyceride (TG) content
  3. 47 Perform Oil Red O (ORO) staining
  4. 48 Quantify ORO-stained lipid content
  5. 49 Perform isopropanol washing for PS visualization
  6. 50 Measure free fatty acid (NEFA) content
  7. 51 Measure interleukin-6 secretion in lipid-loaded organoids
Statistical Analysis and Data Integration Post-experimental data collection
  1. 52 Organize experimental data
  2. 53 Perform one-way ANOVA analysis
  3. 54 Perform paired t-test for BPA level comparison
  4. 55 Analyze PS and BPA interactive effects using GLM
  5. 56 Generate dose-response curves and interaction plots
  6. 57 Synthesize and interpret all molecular and cellular data
  7. 58 Map findings to adverse outcome pathways (AOPs)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Cheng et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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