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LUNG Publication-derived

Lung Organoids for Infection Studies with Immune Cells

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

Authors: Joana Duarte Catarino Duration: 24 days 8 phases Cell source: 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

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

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Attribution

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

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