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LIVER Publication-derived

Mucosal-associated Invariant T Cells Promote Ductular Reaction through Amphiregulin in Biliary Atresia

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

👤 Man-Huan Xiao, Sihan Wu, Peishi Liang, Dong Ma, Jiang Zhang, Huadong Chen, Zhihai Zhong, Juncheng Liu, Hong Jiang, Xuyang Feng, Zhenhua Luo ⏱ 60 days 📋 13 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Xiao et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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