Stem cell-based lung-on-chip models
Inventors
Nawroth, Janna • Barrile, Riccardo • Conegliano, David • Villenave, Remi • Lucchesi, Carolina • Nguyen, Justin • Varone, Antonio • Karalis, Catherine • Hamilton, Geraldine
Assignees
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Publication Number
US-12098352-B2
Publication Date
2024-09-24
Expiration Date
Abstract
An in vitro microfluidic “organ-on-chip” device is described herein that mimics the structure and at least one function of specific areas of the epithelial system in vivo. In particular, a stem cell-based Lung-on-Chip is described. This in vitro microfluidic system can be used for modeling differentiation of cells on-chip into lung cells, e.g., a lung (Lung-On-Chip), bronchial (Airway-On-Chip; small-Airway-On-Chip), alveolar sac (Alveolar-On-Chip), etc., for use in modeling disease states of derived tissue, i.e. as healthy, pre-disease and diseased tissues. Additionally, stem cells under differentiation protocols for deriving (producing) differentiated lung cells off-chips may be seeded onto microfluidic devices at any desired point during the in vitro differentiation pathway for further differentiation on-chip or placed on-chip before, during or after terminal differentiation.
Core Innovation
The invention describes stem cell-based lung organ-on-chip models that include lung, airway, and alveolar configurations using microfluidic devices with epithelial differentiation on-chip. An open-top, stretchable Alveolus-Chip architecture is described in which a porous ECM-coated membrane separates an epithelial/stroma compartment from a spiral endothelial channel, with laminar perfusion in the spiral channel and the alveolar epithelium exposed via an air-liquid interface (ALI).
Co-culture of epithelial cells with fibroblasts and perfused endothelial cells supports long-term viability and differentiated pneumocyte phenotypes including Type I/Type II markers. Surfactant protein expression and secretion are supported, including SP-B detectable by antibody staining and measurable effluent surfactant C, and fibroblasts and cyclic membrane stretching enhance alveolar gene/protein markers and surfactant production.
The invention further describes modeling inflammatory/toxic injury and pharmacology readouts using stimuli such as LPS and H2O2, including Nrf2-pathway activation. The document includes readouts such as barrier leakage and cytokine responses, validates vascular function via blood perfusion/platelet aggregation, and describes a universal ALI culture medium (ALI-M/M199) supporting multiple co-cultured primary cell types and cell source options including primary adult, organoids/spheroids, and hESC/iPSC-derived progenitors expressing markers such as SOX17+/SOX9+/SOX2+/NKX2-1+.
The invention provides a microfluidic device comprising functional Type II lung parenchyma cells and a stroma area or layer comprising fibroblast cells. The functional Type II lung parenchyma cells are cultured together with the fibroblast cells such that the functional Type II lung parenchyma cells secrete surfactant C in amounts between 30-100 ng/ml, with surfactant C secreted daily from day 9 to day 15 of culture.
Claims Coverage
The provided independent claims cover microfluidic culturing and differentiation workflows that produce functional Type II lung parenchyma cells secreting surfactant C in a defined daily range. Across the independent claims, the key inventive features are microfluidic culture with fibroblast-containing stroma, co-culture that increases surfactant C secretion relative to culture without fibroblasts, and differentiation of cells positioned on a microfluidic surface under differentiation conditions.
Microfluidic culture with functional Type II lung parenchyma and fibroblast-containing stroma
A method providing a microfluidic device comprising functional Type II lung parenchyma cells and a stroma area or layer comprising fibroblast cells, and culturing the functional Type II lung parenchyma cells and the fibroblast cells such that the functional Type II lung parenchyma cells secrete surfactant C in amounts between 30-100 ng/ml, wherein the amounts are secreted daily from day 9 to day 15 of culture.
Microfluidic co-culture with fibroblasts increases surfactant C secretion
A method providing a microfluidic device comprising functional Type II lung parenchyma cells and a stroma area or layer comprising fibroblast cells, and culturing the functional Type II lung parenchyma cells and the fibroblast cells such that surfactant C is secreted in amounts greater than where the functional Type II lung parenchyma cells are cultured in the absence of said fibroblast cells, with the amounts secreted daily from day 9 to day 15 of culture after introducing the functional Type II lung parenchyma cells into the microfluidic device.
Differentiation of cells positioned on a microfluidic device surface to functional Type II lung parenchyma
A method providing a microfluidic device comprising a surface and a population of living cells, wherein at least a portion of the living cells have the capability to differentiate into functional Type II lung parenchyma cells, introducing the living cells into the microfluidic device such that the living cells are positioned on the surface, and exposing the positioned cells to conditions that cause at least a portion of the positioned cells to differentiate into functional Type II lung parenchyma cells secreting surfactant C in amounts between 30-100 ng/ml, wherein the amounts are secreted daily from day 9 to day 15 of culture after introducing the living cells into the microfluidic device.
The independent claims collectively define microfluidic methods that generate or maintain functional Type II lung parenchyma cells with fibroblast involvement and differentiation conditions, achieving surfactant C secretion in a daily 30-100 ng/ml range from day 9 to day 15. One independent claim further requires greater surfactant C secretion than culture without fibroblasts, and another independent claim includes positioning living cells on a microfluidic surface followed by differentiation into functional Type II lung parenchyma with the defined surfactant C secretion profile.
Stated Advantages
Supports long-term viability and differentiated pneumocyte phenotypes including Type I/Type II markers.
Supports surfactant protein expression and secretion, including detectable SP-B and measurable effluent surfactant C.
Fibroblasts and cyclic membrane stretching enhance alveolar gene/protein markers and surfactant production.
Enables modeling inflammatory/toxic injury and pharmacology readouts with barrier leakage and cytokine responses.
Validates vascular function via blood perfusion/platelet aggregation.
Surfactant C is secreted in amounts between 30-100 ng/ml daily from day 9 to day 15 of culture.
Surfactant C is secreted in an amount greater than where the functional Type II lung parenchyma cells are cultured in the absence of fibroblast cells.
Documented Applications
Modeling inflammatory/toxic injury and pharmacology readouts using stimuli such as LPS and H2O2, including Nrf2-pathway activation, with barrier leakage and cytokine responses.
Validation of vascular function via blood perfusion/platelet aggregation.
On-chip lung, airway, and alveolar organ-on-chip modeling using microfluidic devices and ALI differentiation workflows.
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