Journal: bioRxiv

Article Title: Membrane-Free Alveolus-on-a-Chip via Biodegradable Scaffold Recapitulates Interstitial Mechanics, Immune Trafficking, and Aerosolized mRNA Delivery

doi: 10.64898/2026.04.17.719302

Figure Lengend Snippet: (A) Schematic illustration of the alveolar microenvironment. The pulmonary alveolus has an average diameter of approximately 200 μm and consists of an epithelial layer lining the air interface and an interstitial compartment composed of extracellular matrix (ECM) populated with lung fibroblasts. (B) Conceptual design of a membrane-free alveolus-on-a-chip platform based on a biodegradable membrane. The curved biodegradable membrane enables reconstruction of an alveolus-like architecture and supports the formation of a biomimetic alveolar microenvironment. (C) Photograph of the assembled chip showing the porous membrane integrated between the apical and basolateral chambers. SEM image showing dome-shaped alveolar microstructures formed on the porous biodegradable membrane. Scale bar: 200 μm.

Article Snippet: The fibroblasts were cultured in Fibroblast Basal Medium (ATCC, PCS-201-030) supplemented with the Fibroblast Growth Kit (ATCC, PCS-201-041) under liquid–liquid culture (LLC) conditions at 37 °C in a humidified atmosphere containing 5% CO2 for up to 3 days until a confluent monolayer was formed.

Techniques: Membrane

Journal: bioRxiv

Article Title: Membrane-Free Alveolus-on-a-Chip via Biodegradable Scaffold Recapitulates Interstitial Mechanics, Immune Trafficking, and Aerosolized mRNA Delivery

doi: 10.64898/2026.04.17.719302

Figure Lengend Snippet: (A) Immunofluorescence image of lung fibroblasts cultured for 7 days on FITC-labeled alveoli-mimetic PLGA membranes, stained with TE-7 (red). Scale bar: 200 μm. ( B) Immunofluorescence image of lung fibroblasts cultured for 7 days on FITC-labeled alveoli-mimetic PLGA membranes, stained with Collagen I (red). Scale bar: 200 μm. (C) SEM image of the lung fibroblast layer cultured for 7 days on the PLGA membrane, viewed from the basolateral side. Scale bar: 50 μm. (D) Raman spectra correspond to the cell-only region (blue), membrane-retained region (red), and pure PLGA (black). (E) Live/dead staining images of lung fibroblasts cultured on the chip at day 1 and day 7. Dashed circles indicate the alveolar structures. Scale bar: 200 μm. (F) Quantification of cell viability of lung fibroblasts cultured on the chip at day 1 and day 7 (n = 4). (G) Side-view immunofluorescence image of lung fibroblasts cultured for 7 days on the chip, stained for fibronectin (red) and collagen I (green). Scale bar: 30 μm. (H) Immunofluorescence images of the 7-day cultured lung fibroblast layer on the PLGA membrane, stained for fibronectin (red), collagen I (green), and DAPI (blue) (top). Following decellularization (bottom), cellular components were removed while the fibroblast-derived ECM network was preserved. Scale bars: 50 μm. (I) Low-magnification SEM image showing lung fibroblasts and the fibroblast-derived ECM cultured for 7 days on the chip (top), and a high-magnification SEM image highlighting the ultrastructure of the ECM network (bottom). Scale bars: 10 μm.

Article Snippet: The fibroblasts were cultured in Fibroblast Basal Medium (ATCC, PCS-201-030) supplemented with the Fibroblast Growth Kit (ATCC, PCS-201-041) under liquid–liquid culture (LLC) conditions at 37 °C in a humidified atmosphere containing 5% CO2 for up to 3 days until a confluent monolayer was formed.

Techniques: Immunofluorescence, Cell Culture, Labeling, Staining, Membrane, Derivative Assay

Journal: bioRxiv

Article Title: Membrane-Free Alveolus-on-a-Chip via Biodegradable Scaffold Recapitulates Interstitial Mechanics, Immune Trafficking, and Aerosolized mRNA Delivery

doi: 10.64898/2026.04.17.719302

Figure Lengend Snippet: (A) Schematic illustration of the co-culture procedure for establishing the alveolar epithelial–fibroblast model on the alveoli-mimetic PLGA membrane. Lung fibroblasts were first seeded on the membrane (day 1), followed by seeding of A549 epithelial cells (day 3). On day 5, the apical medium was removed to initiate ALI culture. During the culture period, the PLGA membrane gradually degraded. (B) Immunofluorescence images of lung fibroblasts cultured on the PLGA membrane for 7 days and A549 cells cultured on top of the fibroblast layer for 5 days, including 2 days under ALI conditions. Lung fibroblasts were stained with TE-7, and A549 cells were stained with ZO-1. Scale bar: 200 μm. (C) SEM images of lung fibroblasts and A549 cells co-cultured on the chip for 7 days. The left panel shows a low-magnification top-view image of the co-culture, while the right panel shows a high-magnification image of A549 cells. Scale bars: 50 μm. (D) Cross-sectional SEM images of lung fibroblasts and A549 cells co-cultured on the chip for 7 days. The left panel shows a region where the membrane remains after partial degradation, while the right panel shows a region where the membrane has fully degraded, leaving only the cellular layer. Scale bars: 5 μm. (E) Immunofluorescence images of lung fibroblasts and A549 cells co-cultured on the chip, stained with TE-7 (green) and ZO-1 (red). The bottom panel shows a cross-sectional view highlighting the epithelial and fibroblast layers. Scale bars: 20 μm. (F) Apparent permeability (P app ) of alveoli-mimetic PLGA membranes after 7 days of degradation, measured for acellular membranes, membranes cultured with lung fibroblasts, and membranes co-cultured with lung fibroblasts and A549 cells on the chip (n = 4) (G) Transepithelial electrical resistance (TEER) of lung fibroblasts and A549 cells co-cultured for 7 days on Transwell® membranes and on the chip. (H) Immunofluorescence images of A549 cells cultured on a fibroblast layer on the chip under ALI conditions for 2 days, stained for SPC, LAMP3, and DAPI. Scale bar: 50 μm. Quantification of SPC (I) and LAMP3 (J) fluorescence intensity in A549 cells cultured under four different conditions (24-well plate, Transwell®, PCL membrane, and PLGA membrane) (n = 4).

Article Snippet: The fibroblasts were cultured in Fibroblast Basal Medium (ATCC, PCS-201-030) supplemented with the Fibroblast Growth Kit (ATCC, PCS-201-041) under liquid–liquid culture (LLC) conditions at 37 °C in a humidified atmosphere containing 5% CO2 for up to 3 days until a confluent monolayer was formed.

Techniques: Co-Culture Assay, Membrane, Immunofluorescence, Cell Culture, Staining, Permeability, Fluorescence

Journal: bioRxiv

Article Title: Membrane-Free Alveolus-on-a-Chip via Biodegradable Scaffold Recapitulates Interstitial Mechanics, Immune Trafficking, and Aerosolized mRNA Delivery

doi: 10.64898/2026.04.17.719302

Figure Lengend Snippet: (A) Tensile stress–strain curves of the Transwell® membrane (blue) and the porous PLGA membrane prior to degradation (red). (B) Young’s modulus of the Transwell® membrane and the porous PLGA membrane prior to degradation (n = 4). (C) Immunofluorescence images of α-SMA expression in fibroblasts cultured on Transwell® and PLGA membranes after 7 days. The yellow dashed circles indicate regions where the PLGA membrane has degraded and is no longer present. Scale bar: 50 μm. (D) Quantification of α-SMA fluorescence intensity in fibroblasts cultured on Transwell® and PLGA membranes after 7 days (n = 4). (E) Schematic illustration of co-culture of fibroblasts and epithelial cells on Transwell® and PLGA membranes. In the Transwell® system, fibroblasts undergo differentiation into myofibroblasts. (F) Reactive oxygen species (ROS) levels in co-culture on Transwell® and PLGA chips. Hydrogen peroxide (H₂O₂) concentration was quantified 24 h after medium exchange (n = 4). (G) Live/dead assay images of co-culture on Transwell® and PLGA chips. Dashed circles indicate the alveolar structures. Scale bar: 200 μm. (H) Quantification of the percentage of dead cells in co-culture on Transwell® and PLGA chips (n = 4). (I) Apparent permeability (P app ) of PLGA membranes after 7 days of degradation and Transwell® membranes in co-culture with lung fibroblasts and A549 cells on the chip (n = 4).

Article Snippet: The fibroblasts were cultured in Fibroblast Basal Medium (ATCC, PCS-201-030) supplemented with the Fibroblast Growth Kit (ATCC, PCS-201-041) under liquid–liquid culture (LLC) conditions at 37 °C in a humidified atmosphere containing 5% CO2 for up to 3 days until a confluent monolayer was formed.

Techniques: Membrane, Immunofluorescence, Expressing, Cell Culture, Fluorescence, Co-Culture Assay, Concentration Assay, Live Dead Assay, Permeability

Journal: bioRxiv

Article Title: Membrane-Free Alveolus-on-a-Chip via Biodegradable Scaffold Recapitulates Interstitial Mechanics, Immune Trafficking, and Aerosolized mRNA Delivery

doi: 10.64898/2026.04.17.719302

Figure Lengend Snippet: (A) Schematic illustration of THP-1 monocyte migration across Transwell®, PCL, and PLGA membranes. Migration is restricted in Transwell® and PCL systems but enabled across the PLGA membrane. (B) Quantification of THP-1 cell migration after 24 h across epithelial–fibroblast co-cultures on the PLGA chip with or without MCP-1 (20 ng mL⁻¹), compared with Transwell® and PCL controls (n = 4). (C) Immunofluorescence images of THP-1 cells collected from the basolateral chamber after transmigrating through the fibroblast–epithelial layer. Cells were stained for CD14 (green) and F-actin (red), with nuclei counterstained with DAPI (blue). Scale bar: 50 μm. (D) Immunofluorescence images of THP-1 cells differentiated into macrophage-like cells following PMA treatment, stained for CD14 and F-actin (top) and CD68 and F-actin (bottom). Scale bar: 50 μm. (E) SEM image of macrophages on the chip under ALI conditions with fibroblast–epithelial co-culture. Scale bar: 30 μm. (F) Immunofluorescence images of lung fibroblasts, A549 cells, and THP-1–derived macrophages cultured on the alveoli-on-a-chip under ALI conditions. Fibroblasts were stained with TE-7, A549 cells were stained with ZO-1, macrophages were stained with CD68. The bottom panel shows a cross-sectional view highlighting the layered organization of the three cell types on the chip. Scale bars: 30 μm.

Article Snippet: The fibroblasts were cultured in Fibroblast Basal Medium (ATCC, PCS-201-030) supplemented with the Fibroblast Growth Kit (ATCC, PCS-201-041) under liquid–liquid culture (LLC) conditions at 37 °C in a humidified atmosphere containing 5% CO2 for up to 3 days until a confluent monolayer was formed.

Techniques: Migration, Membrane, Immunofluorescence, Staining, Co-Culture Assay, Derivative Assay, Cell Culture

Journal: bioRxiv

Article Title: Membrane-Free Alveolus-on-a-Chip via Biodegradable Scaffold Recapitulates Interstitial Mechanics, Immune Trafficking, and Aerosolized mRNA Delivery

doi: 10.64898/2026.04.17.719302

Figure Lengend Snippet: (A) Schematic illustration of aerosol delivery of MOF nanoparticles to the alveoli-on-a-chip using a nebulizer. MOF nanoparticles (1 µg) were aerosolized and deposited onto the epithelial surface under air–liquid interface conditions and exposed to the chip for 48 h. (B) Live/dead fluorescence images of cells cultured on the chip after aerosol exposure to MOF nanoparticles (1 µg, 48 h) compared with untreated controls. Dashed circles indicate the alveolar structures. Scale bar: 200 μm. (C) Quantification of cell viability of cells cultured on the chip before and after MOF nanoparticle exposure (1 µg, 48 h) (n = 4). (D) Immunofluorescence images showing cellular responses in the alveoli-on-a-chip following nanoparticle delivery in the presence of macrophages. Fibroblasts were stained with TE-7, epithelial tight junctions of A549 cells were labeled with ZO-1, GFP indicates nanoparticle-mediated gene expression, and nuclei were counterstained with DAPI. Images correspond to control, LNP-treated (1 µg, 48 h), and MOF-treated (1 µg, 48 h) conditions. Scale bar: 200 μm. (E) Quantification of the proportion of GFP-positive cells following nanoparticle-mediated delivery in the presence or absence of macrophages (MΦ), comparing LNP and MOF nanoparticle formulations after exposure to nanoparticles (1 µg, 48 h) (n = 4). (F) Immunofluorescence images showing cellular responses in the macrophage-containing alveoli-on-a-chip following MOF treatment (1 µg, 48 h). Fibroblasts were stained with TE-7, epithelial tight junctions of A549 cells were labeled with ZO-1, GFP indicates nanoparticle-mediated gene expression, and nuclei were counterstained with DAPI. Scale bar: 50 μm. (G) Side-view immunofluorescence images showing cellular responses in the macrophage-containing alveoli-on-a-chip following MOF treatment (1 µg, 48 h). Scale bar: 30 μm. (H) Pie chart showing the proportion of MOF-transfected cells in the chip, indicating the relative distribution of fibroblasts and epithelial cells among GFP-positive cells. (I) Cytokine secretion profiles measured in the alveoli-on-a-chip 48 h after MOF nanoparticle exposure (1 µg), including MCP-1, GM-CSF, IL-8, and IL-6 (n = 3).

Article Snippet: The fibroblasts were cultured in Fibroblast Basal Medium (ATCC, PCS-201-030) supplemented with the Fibroblast Growth Kit (ATCC, PCS-201-041) under liquid–liquid culture (LLC) conditions at 37 °C in a humidified atmosphere containing 5% CO2 for up to 3 days until a confluent monolayer was formed.

Techniques: Aerosol, Fluorescence, Cell Culture, Immunofluorescence, Staining, Labeling, Gene Expression, Control, Transfection