Human Distal Lung Organoid Eversion for SARS-CoV-2 Infection
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Solution Overview
Problem
Current methods fail to establish long-term, feeder-free, chemically defined 3D culture systems for human distal lung organoids, particularly for alveolar and basal stem cells, limiting the study of distal lung pathologies and the identification of human distal lung stem cell functions, which are often inferred from mouse studies.
Innovation Solution
Development of a feeder-free, chemically defined culture method for human distal lung organoids derived from adult alveolar epithelial type II and KRT5+ basal cells, allowing for clonal expansion and differentiation into club and ciliated cells, with the ability to model SARS-CoV-2 infection by reversing apical-basal polarity to facilitate apical infection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If conventional culture methods are used for human distal lung cells, then short-term culture is achieved, but long-term self-renewal and expansion capacity is lost
Solution Approach 1:
The patent uses feeder-free chemically defined media containing specific growth factors (EGF, FGF7, FGF10, R-spondin1, Noggin, WNT3A) as intermediaries to support long-term culture of human distal lung organoids. These defined factors replace undefined feeder cell signals, enabling reliable long-term culture while maintaining self-renewal capacity of basal and AT2 cells.
Solution Approach 2:
The patent employs parameter changes in culture conditions, including specific oxygen tension (5% O2), temperature (37°C), and chemically defined media composition, to enable long-term culture. These parameter optimizations allow human distal lung organoids to maintain self-renewal capacity for extended periods without feeder cells.
2Stability of the object's composition
If organoids are cultured with apical surfaces directed inwards, then 3D structure is maintained, but pathogen access to apical receptors is prevented
Solution Approach 1:
The patent applies mechanical disruption to invert the organoid structure, turning apical surfaces that were directed inwards towards the lumen to now face outwards. This inversion allows pathogens like SARS-CoV-2 to access apical receptors (ACE2) on the organoid surface, enabling infection modeling while the organoids recover and reform their 3D structure.
Solution Approach 2:
The patent performs preliminary mechanical disruption of organoids before pathogen exposure to enable pathogen access. After infection, the organoids are allowed to recover and reform their 3D structure, thus the structural integrity is restored after the infection assay is complete.
3Reliability
If feeder cells are used to support distal lung cell culture, then cell survival is improved, but biological characterization and screening utility are limited
Solution Approach 1:
The patent extracts and eliminates feeder cells from the culture system, replacing them with chemically defined media containing specific growth factors. This removal of undefined feeder cell components enables full biological characterization of distal lung cells and improves screening utility, while the defined media factors maintain cell survival and self-renewal.
Solution Approach 2:
The patent enables distal lung organoids to self-maintain through chemically defined media factors that support self-renewal and differentiation without external feeder cell support. The system becomes self-sufficient, allowing comprehensive biological characterization and manipulation for research and screening purposes.
Data Source
AI summary
Abstract: We describe a robust human adult distal lung organoid method with a procedure for everting organoids to essentially turn them inside out. This then relocates the apical ACE2-expressing surfaces of cells to the organoid exterior, where they can then be easily infected by SARS-CoV-2 added to the tissue culture medium. Further, this method can be used for infection of any distal lung pathogen that infects apically. Alternatively, if a pathogen interacts basolaterally then eversion is not necessary, and the human adult distal lung organoids can be infected as is.