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LUNG Publication-derived

Modelling BCG Vaccination in Human Lung Organoids: A Hybrid Organoid Platform for Studying Trained Immunity

Source Catarino, 2024 · Center for Biomedical Research of Aragón, University of Zaragoza

👤 Joana Duarte Catarino ⏱ 24 days 📋 8 phases 🧫 Human lung tissue (adult tissue from tumour resection surgeries); Human iPSC-derived or PBMC-derived macrophages and NK cells

Abstract

This protocol establishes a hybrid human lung organoid model containing lung epithelial organoids, innate immune cells (macrophages and natural killer cells), and fibroblasts to study BCG vaccination-induced trained immunity. The protocol optimizes macrophage infection parameters (MOI, incubation duration, antibiotic selection) using fast-growing Mycobacterium smegmatis as a model organism, then validates results with slow-growing BCG Pasteur GFP in the complete 3D organoid assembly.

Cell source
Human lung tissue (adult tissue from tumour resection surgeries); Human iPSC-derived or PBMC-derived macrophages and NK cells
Application
Disease modeling; Vaccination response and trained immunity characterization

Protocol overview

40 steps across 8 phases

Organoid Generation and Maintenance Pre-experimental: ongoing culture
  1. 1 Obtain and Process Human Lung Tissue Samples
  2. 2 Generate Lung Organoids from Tissue Samples
  3. 3 Monitor Organoid Growth and Passage Schedule
  4. 4 Perform Organoid Passage
Immune and Stromal Cell Isolation and Preparation Before assembly into 3D hybrid model
  1. 1 Isolate Peripheral Blood Mononuclear Cells (PBMCs)
  2. 2 Freeze PBMCs for Long-Term Storage
  3. 3 Perform Magnetic-Activated Cell Sorting (MACS) to Isolate Monocytes
  4. 4 Differentiate Monocytes into Macrophages (2D Culture)
Mycobacterial Strain Preparation and Characterization Pre-infection setup (2-20 days depending on strain growth)
  1. 1 Culture Mycobacterial Strains in Standard Liquid Medium
  2. 2 Measure Bacterial Growth by Optical Density
  3. 3 Determine Colony Forming Units (CFU) by Plating
  4. 4 Transform Mycobacteria with GFP Plasmid (Optional)
  5. 5 Confirm GFP Transformation by PCR and Gel Electrophoresis
Antibiotic Susceptibility Testing (Minimum Inhibitory Concentration) Pre-infection (24 hours for Msmeg; 7 days for slow-growing strains)
  1. 1 Prepare MIC Assay Plate
  2. 2 Inoculate Wells with Mycobacteria
  3. 3 Incubate MIC Plate
  4. 4 Add Redox Indicator and Read Plate
Optimization of Macrophage Infection (2D Culture) 8–9 days (5 days differentiation + 1 day inoculation + 1 day post-infection sampling + 7 days culture for CFU determination)
  1. 1 Prepare Mycobacterial Inoculum
  2. 2 Inoculate Macrophages
  3. 3 Wash and Apply First Antibiotic Treatment
  4. 4 Apply Second Antibiotic Treatment and Prolonged Culture
  5. 5 Collect Samples for CFU Count
  6. 6 Collect Samples for Microscopy
Organoid Characterization by Histology and Immunofluorescence Variable (after organoid generation and passage)
  1. 1 Fix and Embed Organoids in Paraffin
  2. 2 Cut Thin Sections with Microtome
  3. 3 Perform Hematoxylin & Eosin (H&E) Staining
  4. 4 Perform Immunofluorescence Staining (Anti-CD14 for Macrophages)
Optimization of 3D Organoid Assembly and Macrophage Infection Efficiency 10–14 days (5 days macrophage differentiation + 1 day inoculation + 1 day for initial sampling + 7 days for final sampling)
  1. 1 Prepare Conditions M (2D Macrophages Only)
  2. 2 Prepare Condition O+M (3D Organoids + Macrophages, Simultaneous Assembly)
  3. 3 Prepare Condition O+iM (3D Organoids + Pre-Infected Macrophages)
  4. 4 Prepare Mycobacterial Inoculum for 3D Infection
  5. 5 Inoculate All Three Conditions
  6. 6 Sample Conditions on Day 0 and Day 7
  7. 7 Refresh Media and Continue Culture
Final Infection of Complete Hybrid Lung Organoid Model with BCG Pasteur GFP 14–19 days (5 days organoid generation/passaging + 5 days macrophage differentiation + 1 day inoculation + day 0, 7, 14 sampling)
  1. 1 Assemble Complete Hybrid Organoid Model
  2. 2 Prepare BCG Pasteur GFP Inoculum
  3. 3 Inoculate Hybrid Organoid Model with BCG Pasteur GFP
  4. 4 Apply Optimized Antibiotic Treatment (Scheme C, Modified)
  5. 5 Collect Samples on Day 0, 7, and 14
  6. 6 Extract RNA for Trained Immunity Marker Analysis

Full SOP

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Attribution

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

This wording is awaiting legal review.

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