[23-2 KIST Europe] Lung Alveolar Organoid with Hybrid Culturing
Component of Lung Epithelium
① Airway Epithelium
including trachea(기관), bronchi(기관지), bronchiole(세기관지)
② Alveolar Epithelium
Culturing Method
Air-liquid Interface (ALI) Culturing
In vitro inhalation model
Combined with an air-liquid exposure system to aerosolize a substance or chemical of interest
ALI Culturing vs 3D Culturing
2D Air-liquid Interface (ALI) Culturing | 3D Culturing | |
PRO | Physiological Relevance Closely mimics the natural environment of alveolar cells in the lungs (inhalation model) Improved Cell Differentiation & Function including surfactant production and proper barrier function for alveolar cells |
Cell-Cell & Cell-Matrix Interactions Allow for complex interactions Longer-Term Culturing support the growth of multiple cell types, reflecting the cellular diversity |
CON | Limited Cell Expansion Since finite lifespan, undergoes only a limited # of cell divisions before stopping proliferating; Limited differentiation into AT1 cells Lengthy Differentiation ALI cultures often grow slowly compared to other in vitro culture systems |
Lack of Mimicking in Vivo Environment Alveolar cells are exposed to air on one side and fluid on the other |
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Motivation for the research
The existing cell organoid culture protocol had the drawback of limited differentiation into AT1 cells which hindered effective gas exchange when using the ALI method, and has an in vivo similarity issue when the alveolar organoid is cultured in the 3D method. To address these issues, an effort was made to create a protocol that integrates the strengths of both ALI culture and 3D culture.
Prior Research & Application
Article | Result | Apply |
(1) | Culture alveolar organoid using hiPSC in a submerged way for 24 days, and maintain it in ALI for 3 days | Use as the main protocol of culturing alveolar organoids with ALI |
(2) | Approximating in-vivo environment, necessary signals supplied by fibroblast-derived factors, Possible to maintain max 120 days | Co-culture with fibroblast for longer maintenance period, efficient differentiation |
(3) | R-spondin2 protein, Amlexanox role as an activator of the canonical WNT/β-Catenin signaling pathway | Add R-spondin2 protein, and Amlexanox in a culture medium |
(4) | Dissociation of 3D spheres organoid into 2D air-liquid culturing, maintained 5-14 days | Introduce methodology into the main protocol |
(1) In vitro modeling of alveolar repair at the air-liquid interface using alveolar epithelial cells derived from hiPSC, Riet S, et al. (2020)
(2) Long-term expansion of alveolar stem cells derived from human iPS cells in organoids, Yamamoto Y, et al. (2017)
(3) A drug screen with approved compounds identifies Amlexanox as a novel Wnt/β-catenin activator, Costa R, et al (2021)
(4) Generating 3D Spheres and 2D Air-Liquid Interface Cultures of hiPSC-derived Type 2 Alveolar Epithelial Cells, Werder R, et al. (2022)
Background
① Fibroblast
Ubiquitous presence; Support, healing, and interaction with immune cell
By co-culturing alveolar organoids with fibroblast, it can activate Paracrine Signaling, Extracellular Matrix (ECM) Remodeling, TGF-β Signaling Pathway, Notch Signaling Pathway, WNT Signaling Pathway, Hypoxia-Induced Factors, … (which has way more effect than just using growth factor in the existing protocol)
② R-spondin2
Consists of secreted proteins known for their role as an activator of the canonical WNT/β-Catenin signaling pathway interacting with LGR4-6 receptors (LGR4: airway, LGR5: branching points of the airway, LGR6: distal airways & alveoli)
Effect on the lung: Lung Development and Regeneration, Stem/Progenitor Cell Regulation especially club cells and AT2 cells, and Interaction with the WNT/β-Catenin signaling pathway
③ Amlexanox
④ WNT Signaling
Role: Early Lung Development; Regulation of Gene Expression, Lung Epithelial Cell Differentiation (AT2 → AT1), Stem Cell Maintenance; Repair and Regeneration, and Interaction with Other Signaling Pathways
Mechanism: Canonical (β-Catenin-Dependent) Pathway regulates gene expression related to cell fate, and Non-Canonical Pathways regulate cell movement and organization
Hybrid Culturing in Alveolar Organoid