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LIVER Publication-derived

Biliary Atresia Models

Full SOP with Academic or a plan

Source Xiao et al., 2024 · Department of Pediatric Surgery and Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China · 10.1016/j.ebiom.2024.105138

Authors: Man-Huan Xiao, Sihan Wu, Peishi Liang, Dong Ma, Jiang Zhang, Huadong Chen, Zhihai Zhong, Juncheng Liu, Hong Jiang, Xuyang Feng, Zhenhua Luo Duration: 60 days 13 phases Cell source: Primary human hepatic mononuclear cells (LMNC) and peripheral blood mononuclear cells (PBMC) from biliary atresia patients; human biliary organoids from hepatoblastoma patients; human cholangiocyte H69 cell line

Abstract

This protocol investigates the role of mucosal-associated invariant T (MAIT) cells in biliary atresia through bulk RNA sequencing, flow cytometry, immunofluorescence staining, and functional co-culture experiments using human liver specimens, biliary organoids, and RRV-infected cholangiocyte-PBMC systems. The study demonstrates that MAIT cells exhibit a wound-healing signature and promote ductular reaction via amphiregulin (AREG) production in a TCR-dependent manner, correlating with advanced fibrosis and poor survival.

Cell source
Primary human hepatic mononuclear cells (LMNC) and peripheral blood mononuclear cells (PBMC) from biliary atresia patients; human biliary organoids from hepatoblastoma patients; human cholangiocyte H69 cell line
Application
Disease modeling and mechanistic investigation of MAIT cell dysfunction in biliary atresia

Protocol overview

54 steps across 13 phases

Phase 1 · Sample Collection and Cell Isolation Day 0-1
  1. a Liver Sample Procurement
  2. b Hepatic Mononuclear Cell (LMNC) Isolation
  3. c Peripheral Blood Mononuclear Cell (PBMC) Isolation
  4. d Cell Culture Media Preparation
Phase 2 · Flow Cytometry Analysis and MAIT Cell Sorting Day 1-3
  1. a Flow Cytometry Staining for MAIT Cell Identification
  2. b Flow Cytometry Analysis
  3. c MAIT Cell Enrichment by Fluorescence-Activated Cell Sorting (FACS)
Phase 3 · In Vitro Stimulation and Intracellular Cytokine Detection Day 1-2
  1. a Intracellular Cytokine Detection via PMA/Ionomycin Stimulation
  2. b IL-12 and IL-18 Cytokine Stimulation
Phase 4 · Immunofluorescence Staining and Tissue Localization Day 1-3
  1. a Immunofluorescence Staining on Frozen Sections
  2. b Image Acquisition and Analysis
Phase 5 · RRV Infection and Co-culture System Establishment Day 1-4
  1. a RRV Virus Preparation
  2. b H69 Cholangiocyte Cell Infection
  3. c Post-infection Cell Washing and PBMC Co-culture
  4. d CD45+ Cell Sorting and Analysis
Phase 6 · Wound-Healing Assay for Cholangiocyte Migration Day 2-3
  1. a H69 Cell Culture and Wound Preparation
  2. b Wound Creation and Media Exchange
  3. c Time-Lapse Imaging
  4. d AREG Blocking Experiment
  5. e Wound Area Quantification
Phase 7 · Cell Proliferation Assay (CCK8) Day 2-3
  1. a H69 Cell Seeding
  2. b MAIT Cell Co-culture or Cytokine Addition
  3. c AREG Blocking (Optional)
  4. d Cell Viability Assessment
Phase 8 · Biliary Organoid Culture and Co-culture Day 0-7
  1. a Biliary Organoid Derivation and Culture
  2. b Organoid Dissociation
  3. c Matrigel Solidification
  4. d Expansion Medium Addition
  5. e MAIT Cell Co-culture
  6. f Organoid Growth Assessment
Phase 9 · Biliary Organoid–LX-2–MAIT Cell Multicellular System Day 0-10
  1. a Biliary Organoid and LX-2 Cell Preparation
  2. b Matrigel Embedding
  3. c Pre-culture Without Inhibitors
  4. d MAIT Cell Co-culture
  5. e Gene Expression Analysis
Phase 10 · RNA Sequencing and Molecular Analysis Day 2-30
  1. a MAIT Cell Enrichment for RNA Extraction
  2. b Total RNA Extraction
  3. c Library Preparation for Bulk RNA-Seq
  4. d Bulk RNA Sequencing
  5. e RNA-Seq Data Analysis
Phase 11 · Single Cell RNA Sequencing (scRNA-seq) Day 5-40
  1. a CD45+ Cell Isolation from RRV Co-culture
  2. b scRNA-seq Library Preparation
  3. c Sequencing on Illumina NovaSeq 6000
  4. d Data Demultiplexing and Preprocessing
  5. e Quality Control and Cell Type Annotation
  6. f Differential Expression and Gene Set Enrichment Analysis
Phase 12 · Intracellular Staining for Flow Cytometry (Cytokine Analysis) Day 3-4
  1. a PBMC Stimulation and Intracellular Staining
  2. b Flow Cytometry Analysis of Intracellular Cytokines
  3. c MR1 Blocking Experiment
Phase 13 · Statistical Analysis and Correlation Studies Day 40-60
  1. a Data Compilation and Organization
  2. b Survival Analysis
  3. c Correlation Analysis
  4. d Gene Expression Correlation Analysis
  5. e Statistical Testing for Group Comparisons

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Attribution

This SOP was written by Organthis based on the published method in Xiao et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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