Microphysiological Models of Lung Epithelium-Alveolar Macrophage Co-Cultures to Study Chronic Lung Disease

Abstract

The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as Chronic Obstructive Pulmonary Disease. In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex three-dimensional architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, we present three complementary microphysiological systems to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, our Transwell system allows the co-culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, our human and mouse precision-cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung-on-a-chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, we provide a complementary approach to better address the intricate mechanisms of lung disease.

ToC figure.

Our MPS platform allows us to recapitulate COPD using our validated chronic cigarette smoke exposure set-up in three complementary systems, namely, co-culture of differentiated lung epithelial cells on ALI with immune effector cells, precision cut lung slices, and a tissue chip coculture model. Diagrams created using BioRender.com.

1. INTRODUCTION

Respiratory diseases are among the most common causes of morbidity and mortality worldwide. Globally, 545 million people were diagnosed with chronic respiratory disease in 2017, an increase of 39.8% since 1990, with most of those cases caused by chronic obstructive pulmonary disease (COPD). ^[1] COPD develops over decades due to chronic exposure to noxious particles or gases, most commonly cigarette smoke (CS). The respiratory system is an essential interface between the body and the outside environment. It comprises the upper airway, which encompasses the nasal cavity, pharynx, and larynx, and the lower airway, which contains the trachea, bronchi, and lungs. The lower airway leads to the alveoli, which exchange oxygen between the lungs and blood and produce surfactant. ^[2–3]

Epithelial cells in the lung interface directly with inhaled air, and many in vitro models focus on modeling the airway or alveolar epithelium. Our group has demonstrated that in response to chronic harmful stimuli in the inhaled air, such as cigarette smoke, epithelial injury drives tissue remodeling seen in COPD.^[4–6] In previous models of lung epithelium, cells are grown at an air-liquid interface (ALI) with media in the basal chamber and the apical cell surface exposed to air, which is a more physiologically accurate model compared to traditional models where cells are entirely submerged.^[7] However, even here, the airway epithelia are typically grown as monocultures on Transwell inserts. These monocultures do not account for the complex milieu in which the epithelial cells interact with immune effector cells and other structural components such as the extracellular matrix (ECM). This interaction allows for the molecular crosstalk mediated by biochemical and physical signals influencing epithelial function. When studying immunity in barrier properties, physiologically relevant models of the lungs need to have the ability to mimic the presence of local immune cells, such as alveolar macrophages present in the lumen of the airways or interstitial macrophages located underneath the epithelium. ^[8] The diversity in both structure and function of different parts of the pulmonary system, such as the airway and alveolar epithelia, also creates challenges when trying to recapitulate them with in vitro models.

The pseudostratified airway epithelium comprises basal, goblet, ciliated columnar, undifferentiated basal, and other secretory cells. Goblet cells produce airway mucus that traps particles expelled by ciliated columnar cells.^[9] These multi-ciliated cells orient by planar cell polarity signaling, a two-step process in which cell-to-cell communication via protein complexes aligns the epithelial cells to the proximal-distal tissue axis, then they organize their cilia in the proximal direction.^[10] Throughout the lung, the maintenance of cellular polarity and tissue architectures is essential for the barrier function of the epithelium.^[11] The airway terminates in the alveoli, a hollow octahedral cavity with an average diameter of 200 μm, which stretches during breathing. The alveolar epithelium comprises type 1 (AEI) and 2 (AEII) epithelial cells. AEI cells cover 93% of the alveolar surface, forming tight barriers with junctional complexes.

AEII reside in “corner-like” areas protected from mechanical stresses and produce surfactant, a mixture of lipids and proteins that reduces the surface tension of the air-liquid interface within the alveoli to minimize the effort needed for breathing and prevent alveolar collapse during expiration. AEII can also proliferate and differentiate into AEI cells, making them essential to alveolar repair^[12]. The alveolar basement membrane supports the epithelial cells and comprises collagen, elastin, proteoglycans, and other extracellular matrix proteins (ECM).^[13] The traditional in vitro barrier tissue models cannot recapitulate these differences in epithelial tissues of the pulmonary system. Therefore, more complex microphysological systems are required to study pulmonary epithelial cell function and disease mechanisms effectively.

Immune cells play a critical role within the airway and alveolar lumen due to the exposure of this body area to the external environment and are necessary to maintain lung homeostasis. Alveolar macrophages clear environmental particulates, pathogens, and apoptotic cells in the lungs without disrupting essential functions such as mucus production and gas exchange.^[14] These macrophages typically derive from immune progenitors in the lungs. They can “self-renew,” although previous literature has indicated that monocytes from circulating blood can be recruited to the lungs and will differentiate into an alveolar macrophage phenotype in response to injury. ^[14–16] Macrophages interact with the epithelium via cell surface receptors and cytokines. They can develop different phenotypes to attack pathogens and regulate other immune effectors by modulating adaptive immune responses. Alveolar macrophages are typically classified by the expression of specific surface proteins. ^[16–18] These phenotypic characteristics can dictate the two major and opposing activities of macrophages, with the M1 macrophages providing a pro-inflammatory role and the M2 macrophages being immunosuppressive. Both external stimuli, such as infection, and internal stimuli, such as signals from other local cells, can drive the polarization of the macrophage.^[17] The complex environment of the pulmonary system requires different approaches and designs to answer specific questions about epithelial cell mechanisms and pulmonary disease. To address this need, we have optimized three microphysiological systems to study the relationship between CS and COPD. First, we have a transwell co-culture model incorporating alveolar macrophages added to either human or mouse cells to study the impact of CS on phenotypic changes in airway epithelium. Next, we utilize human precision-cut lung slices to study the remodeling of the parenchyma in the native structure of the lungs in response to CS. In addition, using mouse precision-cut lung slices co-cultured with alveolar macrophages, we address macrophage dynamics and cell interactions in response to CS. Finally, we designed a membrane-free lung-on-a-chip to model the direct co-culture of human epithelial and immune cells and allow for direct imaging of the cross-section of the pseudostratified, polarized airway epithelium. Each of these approaches offers unique advantages and functional outputs that can contribute to a broader understanding of the pulmonary epithelium in the context of COPD.

2. MATERIALS AND METHODS

2.1. Animal protocol

All animal experiments and procedures were performed under the approval and guidelines of The Johns Hopkins University Animal Care and Use Program.

2.2. Airway epithelial and immune co-culture

In the mouse model, airway epithelial cells, alveolar macrophages, and peripheral blood mononuclear cells were isolated from age- and sex-matched FVB/NJ mice older than four weeks. Mice were sacrificed in a sealed CO2 exposure chamber and subsequent cervical dislocation.

Whole blood was collected by cardiac puncture at room temperature, and mouse PBMCs were isolated via centrifugation of whole blood. Subsequently, the trachea of the mice was exposed, and a catheter was placed inside the trachea to instill buffer with calcium and magnesium free DPBS (ThermoFisher), 1 mM Ethylenediaminetetraacetic acid, EDTA) to isolate alveolar macrophages. The lavage buffer was instilled intratracheally with a 1 mL syringe, and the 1mL buffer was gently instilled and withdrawn three to five times before being collected in an aliquot of macrophage media (RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin, and 1mM L-glutamine). Subsequently, the tracheas were removed and placed in a pronase solution for 16 hours at 4°C. Cells were isolated with a 70 μm cell strainer, resuspended in DMEM with 10% FBS and incubated on a T-75 tissue culture flask for 4 hours to allow macrophages and fibroblasts to adhere to the surface. After incubation, the non-adherent basal airway epithelial cells were resuspended in Ex Plus media (Stemcell) prepared to manufacturer specification on a collagen-coated T-75 flask to allow the basal cells to adhere and proliferate. For normal human bronchial epithelial cells (NHBE), frozen cell pellets sourced from patient bronchial brushings (Lonza) were revived and cultured in collagen-coated T-75 flasks to grow to confluence (Table S1). THP-1 cells (human monocytic cell line) were cultured in suspension with RPMI+L-glutamine+10% FBS, then passaged and differentiated into macrophages in batches of 4×10⁶ cells in 10 cm cell culture dishes with media supplemented with 100 nM phorbol 12-myristate 12-acetate (PMA) for 72 hours. Finally, differentiated THP-1 cells were lifted with 0.25% trypsin and neutralized for co-culture.

Both mouse and human basal epithelial cells were differentiated to mature pseudostratified epithelia when further cultured on an air-liquid interface on collagen-coated Transwells (100,000 cells/cm²) after reaching confluence and switching to ALI (Stemcell) media prepared to manufacturer specification. After switching to air-liquid interface culture, 1×10⁶ macrophages were resuspended in 6 μL macrophage media or RPMI and placed on the apical surface of the Transwell inserts. Transwells were co-cultured after epithelial cells had been fully differentiated, observed by the presence of beating cilia under brightfield microscopy. The Transwell ALI differentiation protocol produces pseudostratified epithelial cells validated by immunostaining of PKC-ζ, and Na+/K+-ATPase described previously. ^[4–5]

2.3. Exposure of cells to cigarette smoke

Cells were exposed to CS or humidified air using our validated smoking chamber, allowing for apical exposure. ^[19] Each smoke exposure consists of two cigarettes that burned for approximately 8 minutes each based on the ISO puff regimen, in which a 35-ml pump is applied every 60 seconds with an 8-second exhaust, one exposure per day. Control inserts experienced the same regimen with humidified air.

The membrane was cut from the Transwell support, mounted, fixed in 4% paraformaldehyde in permeabilized in 0.1% Triton X-100 for immunofluorescence. After permeabilization, the cells were washed with calcium and magnesium-free DPBS (ThermoFisher), and incubated overnight at 4°C. After blocking, the cells were washed with calcium and magnesium-free DPBS (ThermoFisher) and incubated overnight at 4°C in a solution containing primary antibodies for CD11c (Invitrogen, CAT: PA5–79537) and Siglec F (Invitrogen, CAT: 14–1702-82) diluted to manufacture recommendations. Next, the primary antibody solution was washed with calcium and magnesium-free DPBS (ThermoFisher) and incubated with the secondary antibody for two hours at room temperature. After incubation, the slides were incubated with Hoechst stain (ThermoFisher, CAT: 62249) for fifteen minutes at room temperature, washed five times with calcium and magnesium-free DPBS (ThermoFisher), and finally covered with mounting media (ProLong Gold Antifade, Thermofisher, CAT: P36930) and a coverslip for imaging. After overnight recovery, the slides were imaged using a Zeiss LSM700 confocal microscope, and images were analyzed using ImageJ software.

2.4. Fluorescence recovery after photobleaching (FRAP)

For fluorescence recovery after photobleaching (FRAP), inserts were exposed as indicated and then stained with SiR-actin, a fluorogenic derivative of jasplakinolide specific to F-actin. Each insert was cultured with 4 μm of SiR-actin on both the apical and basal sides for 24 hours. After incubation, the inserts were mounted on glass coverslips with the stained media shortly before imaging on a Zeiss LSM700 confocal microscope. Several regions of interest (ROIs) were bleached at full power with a 639 nm wavelength, with an unbleached control area to account for the background loss of fluorescence. The fluorescence recovery was monitored for approximately 10 minutes (ZEN software), and the resulting data were normalized to initial fluorescence values and corrected for background loss of fluorescence. The cells were characterized by measuring the average half-time of fluorescence recovery after bleaching and the mobile fraction of the F-actin within the cells. The mobile fraction was calculated using the formula:

$$Fm=\frac{\left(I_E-I_0\right)}{\left(I_I-I_0\right)}$$

Where the final value of recovered fluorescence determines the mobile fraction (Fm) after bleaching (IE), the first fluorescence intensity after bleaching (I0), and the initial fluorescence intensity before bleaching (II).

2.5. Fluorescence-activated cell sorting (FACS) of alveolar macrophages

For cell sorting, co-cultured mouse cells were dissociated with accutase (StemCell), and replicates were pooled. Then, cells were counted, resuspended in FACS buffer, and incubated with CD11b (Biolegend CAT: 101228), CD11c (BD Biosciences CAT: 565452), I-Ab (Biolegend CAT:116405), and F4/80 fluorophore-conjugated antibodies (Biolegend CAT:1231118) and sorted using the JHU Ross Flow Cytometry Core to evaluate M1/M2 polarization of the macrophages (Figure S1-A–G).

2.6. Human Precision Cut Lung Slices (human PCLS)

Human PCLS from three different normal donors were obtained from the International Institute for the Advancement of Medicine (IIAM) and the Institute for In Vitro Science (IIVS). Seven replicate slices per donor were used for each experimental condition (Table S2). Human PCLS were used only to optimize the CS exposure model using our in-house developed smoking chamber.^[19]

2.7. Exposure of human precision-cut lung slices (human PCLS) to cigarette smoke.

Adjacent human PCLS were exposed to either CS aerosols or humidified air using our in-house developed smoking chamber and as described previously with some modifications. ^[19–23] Each Transwell (Corning CAT:3450) containing the individual human PCLS was transferred to the smoking chamber. Human PCLS culture media (DMEM F12 1:1 (Sigma), supplemented with 1% antibiotic-antimycotic (ThermoFisher Scientific), and 1% insulin- transferrin-selenium (ITS-G, Gibco)) was added only to the basal side of the chamber, and the temperature was maintained at 37°C throughout the exposures. Human PCLS were exposed to a total of eight cigarettes. After exposures, human PCLS were removed from the inserts and placed into a 24-well plate containing human PCLS culture media (1 mL per slice).

Human PCLS were allowed to incubate for 24 hours (at 37°C and 5% CO2). Post the 24-hour incubation, the human PCLS supernatant was collected for LDH assay. The remaining slices were used for the Alamar Blue assay, and TMRM assay, and some were fixed for histology.

2.8. Histology

To generate H&E and IHC (IF) slides, human PCLS were fixed for 24 hours overnight at 4°C immediately after live imaging, using 4% paraformaldehyde (ThermoFisher), and then processed using The Johns Reference Histology Center. H&E slides were scanned by the Johns Hopkins Oncology Tissue Services (SKCCC).

2.9. Mean linear intercept analysis (MLI)

The human PCLS H&E scanned slides were analyzed as described.^[24] Digital images were taken at 10x magnification and cropped at 1000 × 1000 (pixels). 580 horizontal and vertical chords were measured. The average height and width of each image were used for analysis.

2.10. Lactate Dehydrogenase Assay (LDH)

To measure the cytotoxicity and cell death of the CS exposure model in human PCLS, a kinetic LDH assay (Abcam) was performed. The supernatant of each slice was collected after the exposures and processed as described by the manufacturer’s protocol. The LDH activity was measured for 60 minutes in kinetic mode at 450 nm using a microplate reader (BioTek Synergy HTX Multi-Mode Microplate Reader S1LFA).

2.11. Mitochondria Membrane Potential (TMRM Assay)

Human PCLS were made into cell suspensions and processed as described by the manufacturer’s protocol (TMRM assay, Abcam). Fluorescence intensity was measured at Ex/Em 549/575 using a microplate reader (BioTek Synergy HTX Multi-Mode Microplate Reader S1LFA). Data was measured in triplicate values and presented as averages.

2.12. Mouse Precision Cut Lung Slices (PCLS)

Mouse PCLS were used to optimize the alveolar macrophage co-culture plus CS exposure model. Mouse PCLS were generated as described previously.^[20] Briefly, age and sex-matched C57BL/6J mice were euthanized following institutional guidelines. First, an incision was made in the abdominal cavity to isolate the lungs. The lungs were inflated with a 2% low melting point agarose (in HBSS, warmed at 37–40°C) via the trachea using a 21G needle. After inflation, the lungs were excised and placed in a 50 mL conical tube filled with cold HBSS. Excised lungs were allowed to solidify at 4°C for 30 minutes, followed by slicing of the left lung (250 mm) using a vibrating blade vibratome (Microm HM650V, SN:3396). Slices were then placed in a 24-well plate submerged in PCLS culture media (DMEM F12 1:1, Sigma), supplemented with 1% antibiotic-antimycotic (ThermoFisher Scientific), and 1% insulin-transferrin-selenium (ITS-G, Gibco). Slices were allowed to acclimate for 48 hours to allow them to recover post-slicing.^[24] After 48 hours, to check for viability, each slice was incubated overnight (at 37°C and 5% CO2) in 1x Alamar Blue HS reagent (ThermoFisher Scientific) diluted in PCLS culture media. 570 nm and 600 nm absorbance were measured the following day using a microplate reader (BioTek Synergy HTX Multi-Mode Microplate Reader S1LFA). Only viable slices were used for the experiments, and all experiments were performed within 48 hours post-slicing.

2.13. Alveolar macrophages isolation and cell sorting from mouse lungs

Alveolar macrophages were isolated and cultured from age and sex-matched C57BL/6J mice as described previously. ^[25] The mouse left lung was used to generate PCLS, and the remaining lung lobes were dissected and dissociated in Liberase TL (5 U/mL) and DNase I (300 mg/mL). After incubation, the dissociated lungs were lysed using Ammonium–chloride–potassium (ACK) lysed buffer (ThermoFisher) for 15 minutes, then washed and filtered with calcium and magnesium-free DPBS (ThermoFisher) containing 2% FBS (ThermoFisher, Heat Inactivated) and 2 mM EDTA (Corning), using a 100 μm cell strainer. After the washing steps, the cell suspension was centrifuged for 10 minutes at 500 RCF and 4°C. The washing and centrifugation steps were repeated twice.

The cell suspension was washed once with FACS buffer (BD Biosciences) for cell sorting and then incubated in mouse Fc block (10 mg/mL, clone 2.4G2, BD Biosciences) on ice for 20 minutes. After Fc block incubation, the cells were washed once with FACS buffer, with the following antibodies: anti-CD45 mouse (Biolegend CAT:147709), anti-CD11c mouse (BD Biosciences CAT: 565452), anti-CD11b mouse (Biolegend CAT:101228), anti-F4/80 mouse (Biolegend CAT:123118), and PE anti-CD206 mouse (Biolegend CAT: 141706). Cell sorting was performed at the Johns Hopkins University Ross Flow Cytometry Core. CD45-positive/CD11c-positive/CD11b-negative/ F4/80-positive/CD206-positive cells were identified as alveolar macrophages (Figure S2).

After cell sorting, alveolar macrophages were centrifuged at 300 RCF (4°C) for 10 minutes and resuspended into a T25 flask with culture medium (DMEM plus 10% FBS, 2% mouse GM-CSF, 1mM Sodium Pyruvate (Millipore Sigma), 10mM HEPES (Gibco), 1x Penicillin/Streptomycin (ThermoFisher), then incubated at 37°C and 5% CO2 for two days.

2.14. Live immunofluorescence staining of mouse precision-cut lung slices (PCLS)

Prior to CS exposure, PCLS slices were incubated for one hour in PCLS culture media containing Cell Mask Actin Tracking Stain- Green (3 μl/ mL of media, ThermoFisher CAT: A57243) and NucBlue live nuclear stain (2 drops, per mL of media, ThermoFisher CAT: R37605) at 37°C and 5% CO2. After incubation, the cell mask Actin and DAPI stained PCLS were washed three times (5-minute incubation each time) with 1x calcium and magnesium-free DPBS (ThermoFisher), then transferred to 12-well inserts (Corning, CAT:3450).

2.15. Exposure of mouse PCLS to cigarette smoke.

Adjacent PCLS were exposed to either CS aerosols or humidified air using our in-house developed smoking chamber and as described previously with some modifications. ^[19–22] Each Transwell containing the individual PCLS slices was transferred to the smoking chamber. PCLS culture media was added only to the basal side of the chamber, and the temperature was maintained at 37°C throughout the exposures. PCLS were exposed to 8 cigarettes. After exposure, PCLS slices were removed from the inserts and immediately placed into a 35 mm petri dish coated with 5% agarose (one PCLS per dish, with no media added) and placed back in the incubator (37°C and 5% CO2).

2.16. Mouse PCLS and alveolar macrophages co-culture

Immediately following CS exposure, alveolar macrophages were added to the PCLS to infiltrate the tissue. ^[26] Briefly, alveolar macrophages were centrifuged at 300 × g (4°C) for 6 minutes in a 5 mL FACS tube and resuspended in alveolar macrophages culture media containing 2.5 ml/mL of Cell Tracker Deep Red CFMDA (ThermoFisher, CAT: C34565). Alveolar macrophages were allowed to incubate in CFMDA for 30 minutes at 37°C and 5% CO2.

After 30 minutes, alveolar macrophages were centrifuged for 5 minutes at 250 × g (4°C) and resuspended in fresh alveolar macrophage media without CFMDA. Freshly CFMDA-stained macrophages were added to each PCLS slice at equal cell density (3,000 alveolar macrophages/slice). PCLS were allowed to incubate with the CFMDA-stained alveolar macrophages for 1 hour at 37°C and 5% CO2. After 1 hour, the co-culture PLCS + alveolar macrophages were washed three times with calcium and magnesium free DPBS (ThermoFisher) and then transferred to glass bottom 35 mm imaging plates (Ibidi, CAT: 81218–200) containing 0.500 mL of fresh PCLS culture media.

2.17. Live confocal imaging of mouse precision-cut lung slices (PCLS)

Following the alveolar macrophage co-culture, PCLS were taken for imaging performed using the Johns Hopkins University Microscope Facility Zeiss LSM 880 Confocal with Airyscan FASTModule (NIH Grant: S10OD023548).

2.18. Tissue Chip

3D-printed molds were designed in Autodesk and printed in resin using a high-resolution 10 μm series 3D printer (Boston Microfabrication). The design consists of 3 interconnected parallel channels, each 1 mm wide. The central channel is 250 μm tall, while the two connected side channels are 500 μm tall. PDMS (Sylgard 184, DOW) prepolymer was mixed 10:1 with a curing agent and poured over the mold, which was placed in a low-pressure chamber to remove any air from the solution, and then cured overnight at 60 °C. After curing, the PDMS was de-molded and exposed to oxygen plasma for bonding to a glass coverslip. The chip was UV sterilized for 30 minutes, then plasma-treated again, filled with Poly-L-Ornithine solution, and incubated for one hour at room temperature. The chip was then washed with DI water, coated in a 1% Matrigel solution in PBS, and incubated overnight at 37°C. After incubation, the chip was placed on a metal heating block in ice to accommodate working with Matrigel, and a 50:50 Matrigel: media solution was flowed through the central channel with and without differentiated THP-1 macrophages. The smaller space in the central channel compared to the connected side channels confines the Matrigel solution to the channel with surface tension, preventing it from flooding into the side channels before the solution can be polymerized in the incubator (Figure S3). As mentioned previously, the THP-1 cells were cultured separately and differentiated with 100 nM PMA, then ~ 50,000 cells/μL were suspended in 7 μL of equal parts Matrigel:THP-1 media solution and injected into the central channel. The chip was then incubated for fifteen minutes at 37°C to solidify the gel, after which 300,000 NHBE cells were suspended in Ex-plus media and added to the side channel. After the NHBE cells were seeded, Ex-plus media was added to the unused side channel, and the chip was cultured under normal cell culture conditions in a plate resting at a 90° angle for 24 hours to allow the epithelial cells to become confluent and grow along the side of the channel shared with the central channel. Media was changed in both channels every 24 hours, and cell adherence was observed with brightfield microscopy. After the epithelial cells reached confluence, media was removed from the epithelial channel to allow the cells to grow at the air-liquid interface, and media was replaced in the media channel every 24 hours. For the endothelial-seeded chip, the tissue chip was UV-sterilized and plasma treated, then coated with attachment factor solution (Cell Applications) for 30 minutes at 37°C. A solution of equal parts Matrigel: Endothelial Cell Growth Basal Medium-2 (EBM-2, Lonza, prepared to manufacturer’s specifications) was used to form the hydrogel in the central channel as described previously. 500,000 human primary lung microvascular endothelial cells (HPMEC, Cell Biologics, CAT H-6011 were seeded into the media channel of the chip and left to adhere overnight at 37°C in normal cell culture conditions. The endothelial chip was fed with EBM-2 daily in submerged culture and characterized with brightfield microscopy to assess confluence. The epithelial chip was immunostained with anti-E-cadherin (Millipore-Sigma, CAT: MABT26) diluted to manufacturer’s specifications, and NucBlue live nuclear stain (2 drops per mL of media, ThermoFisher CAT: R37605). The endothelial chip was stained with anti-CD31 (Abcam, CAT: ab28364) diluted to the manufacturer’s specifications and NucBlue live nuclear stain.

2.19. Statistical analysis

Statistical analysis was performed using Prism 9. Data were presented as mean ± SEM of each group. For the human and mouse PCLS raw data was analyzed, and no preprocessing (transformation, normalization, removal of outliers) of the data was performed. Data were analyzed with a paired t-test. A p value of ≤ 0.05 was considered significant. For the human Transwell co-cultures, three donors were used for the exposure experiments, with at least three replicate Transwells for each experimental condition. For the murine Transwell co-cultures, four mice were used to generate basal cells for each experiment, and four additional age-matched mice were used to obtain alveolar macrophages for co-culture. After sorting, numbers of M1 and M2 macrophages in each treatment group were normalized to unexposed co-culture M1/M2 cell numbers and presented in a graphical format. All FRAP experiments were completed with 5 bleached regions on Transwells. All FRAP curves were normalized to pre-bleached values and all normalized regions of interest were averaged for each condition, and the standard error was calculated for each time point. The lung-on-a-chip used epithelial cells from 1 donor, for both culture conditions. For the human precision-cut lung slices a total of three different normal donors were used, with seven replicate slices in each experimental group. For the mouse precision-cut lung slices three different mice were used with three replicate slices in each experimental group.

3. RESULTS

Three strategies were used to model the pulmonary system: Transwell co-culture, human precision-cut lung slices, and a membrane-free lung-on-a-chip, which collectively enable the modeling of interactions between human epithelium and immune cells, offering a functional approach to studying the pulmonary epithelium in COPD (Figure 1).

Figure 1.

Microphysiological models of coculture and cigarette smoke of the lung epithelium. (A) Diagram of Transwell-based airway epithelial and alveolar macrophage coculture. (B) Diagram of the 3-compartment lung-on-a-chip model, with a cell-laden gel in the central channel and human bronchial airway epithelial cells growing on the wall between the epithelial and gel compartments to form a barrier. (D) PCLS explant cultures exposed to direct cigarette smoke using our in-house developed smoking chamber. Diagram created using BioRender.com.

3.1. Co-culture with epithelial cells drives PBMCs to have an alveolar macrophage phenotype

As several subpopulations of macrophages are present in the lungs, peripheral blood mononuclear cells (PBMCs) were extracted from whole blood and co-cultured with airway epithelial cells to determine if the epithelial milieu modified the phenotype of the PBMCs (Figure 2). Monocytes from whole blood typically express CD11c on the cell surface but are negative for Siglec F. ^[27] After we co-cultured the epithelium with the PBMCs for seven days, immunostaining demonstrated that the co-culture contained cells expressed both CD11c and Siglec F (Figure 2A, Figure 2B), indicating that with co-culture, the PBMCs adopted an alveolar macrophage phenotype. Monocultured alveolar macrophages stained positively for CD11c and Siglec F (Figure 2C), and PBMCs stained positively for CD11c and negatively for Siglec F (Figure 2D).

Figure 2.

Immunostaining of murine cocultures of epithelial and immune cells. (A) Murine airway epithelial cells cocultured with alveolar macrophages (AMs), scale bar: 70 μm. (B) Murine airway epithelial cells cocultured with PBMCs, scale bar: 25 μm. (C) Control staining of monocultured alveolar macrophages, scale bar: 70 μm. (D) Control staining of PBMCs, which lack Siglec F expression, scale bar: 25 μm. Fluorescently labeled surface markers identify macrophages after 7 days of coculture. Alveolar macrophage markers CD11c (green, left) and Siglec F (red, center) are present in both cocultures, indicating an altered phenotype in the cocultured PBMCs. Merged images (right) are shown with nuclear Hoechst stain (blue).

3.2. Epithelial co-culture is needed to study macrophage polarization after CS injury

We evaluated the effects of CS in the short and long term on macrophage polarization after one week of co-culture of mouse airway epithelial cells and alveolar macrophages using a custom developed smoking chamber (Figure 3). Without epithelial co-culture, the macrophages did not survive even short CS exposures. We sorted the co-cultures of epithelial cells and macrophages to identify macrophage polarization after one and ten days of exposure and after a ten-day recovery period. Cells were sorted for F4/80⁺ /CD11C⁺ / CD206⁻ surface expression to identify M1 pro-inflammatory polarization, while M2 anti-inflammatory polarization was measured by F4/80⁺/CD11C⁻/CD206⁺ surface expression (Table 1, Figure 4A). At baseline, the macrophages had M2 characterization based on surface markers (Figure 4B). All air exposed conditions also had macrophage populations with M2 surface expression (Figure 4A, Figure 4C, Figure 4E, Figure 4G) A short exposure (one day) of CS did not significantly change the macrophage markers, with most still demonstrating an M2 phenotype (Figure 4D). However, prolonged CS exposure (ten days) shifted the macrophages into pro-inflammatory M1 cells (Figure 4F), which reflects in vivo findings.^[28–29] After prolonged recovery, the macrophages reverted to baseline with predominantly M2 macrophages (Figure 4H).

Figure 3.

Graphical representation of the cigarette smoke exposure model optimized using our in-house developed smoking chamber.16 (A) Diagram of the Transwell holding inside the chamber. (B) Image of our device with a PCLS holding Transwell as well as a cell monolayer Transwell. Diagram created using BioRender.com.

Table 1.

Results of macrophage polarization identification by fluorescent activated cell sorting (FACS).

		M1 A	M2 B	Total Cell Count
Untreated C		66	1311	2.14 × 10 6
	Air D	28	1020	1.2 × 10 6
1-day immediate	CS E	23	2538	1.7 × 10 6
	Air F	40	801	1.77 × 10 6
10-day Immediate	CS G	112	2	9.28 × 10 6
	Air H	29	28774	2.5 × 10 6
10-day recovery	CS I	69	10970	3.7 × 10 6

^AM1 (F4/80+, CD11C+, CD206−),

^BM2 (F4/80+, CD11C−, CD206+),

^CFIGURE S1-A,

^DFIGURE S1-B,

^EFIGURE S1-C,

^F FIGURE S1-D

^GFIGURE S1-E,

^HFIGURE S2-F,

^IFIGURE S2-G.

Figure 4.

Identification of murine alveolar macrophage polarization after coculture and exposure to cigarette smoke. (A) Graphical representation of macrophage polarization after coculture and exposure to cigarette smoke as determined by FACS. All numbers of reported M1 and M2 cells are represented by fold change as compared to sorted M1/M2 macrophages in unexposed cocultures. (B) CD11c vs. CD206 expression profiles of unexposed cocultured murine alveolar macrophages. (C) CD11c vs. CD206 expression profiles of 1-day CS and (D) air-exposed cocultured murine alveolar macrophages. (E) CD11c vs. CD206 expression profiles of 10-day CS and (F) air-exposed cocultured murine alveolar macrophages. (G) CD11c vs. CD206 expression profiles of 10-day CS and (H) air-exposed cocultured murine alveolar macrophages after an additional 10 days of recovery.

3.3. Co-culture with macrophages alters epithelial actin dynamics in response to pathologic stimuli

We have shown that prolonged CS injury can increase cortical tension and the polymerized actin in airway epithelial cells. ^[19–22] This increase in polymerized actin was in part reflected by increased stress fiber formation. Still, crosstalk between other cell types in the airways can modulate actin dynamics.

FRAP analysis of stained actin filaments in murine tracheal epithelial cells show that CS causes a decrease in the mobile fraction of actin and an increase in the half-time of recovery (Figure 5A), indicating a higher level of stable, polymerized actin. In mouse cells, we found that adding macrophages reduced the recovery half-time of epithelial actin, indicating increased F-actin turnover in epithelial cells (Figure 5B). As a confirmation in human cells, we found that the addition of macrophages to primary differentiated human epithelia also reduced the recovery half-time of epithelial actin (Figure 5C). To test the role macrophages play in epithelial injury response, we exposed human macrophage-epithelial co-cultures to CS to assess actin turnover with injury. A single CS exposure was sufficient to reduce the mobile fraction of actin and increase the recovery half-time in the human epithelial monoculture (Figure 5D), indicating an increased fraction of polymerized actin with this insult. The addition of macrophages increased the half- time of fluorescence recovery after CS exposure (Figure 5F), indicating that the macrophages increase epithelial actin turnover to potentially reduce stress fiber formation. However, this ability is diminished after prolonged CS exposure, where the epithelial mobile fraction of actin is reduced with increased recovery half-time despite the presence of macrophages (Figure 5E).

Figure 5.

Fluorescent labeling of F-actin in Transwell models of human and murine airway epithelial cells and macrophage coculture. (A) Fluorescence recovery of murine airway epithelial cells and alveolar macrophages exposed to 10 days of air or cigarette smoke. (B) Fluorescence recovery of murine epithelial cells with added alveolar macrophages. (C) Fluorescence recovery of human airway epithelial cells with added differentiated THP-1 macrophages had a similar increase in recovery in response to coculture. (D) Fluorescence recovery of human airway epithelial cells coculture with differentiated THP-1 macrophages. (E) Human airway bronchial epithelial cells and differentiated THP-1 macrophages exposed to 1 day of CS. (F) Human airway bronchial epithelial cells and differentiated THP-1 macrophages were exposed to 10 days of CS 10-day of CS. Error bars = SE, n=5.

3.4. Using precision-cut lung slices to study the effects of cigarette smoke

Using human precision-cut lung slices and our in-house smoking chamber, we optimized cigarette smoke exposure to study cells in their in vivo organization and native environment.^[19–20] This enables us to study the progressive tissue remodeling that occurs over time in response to cigarette smoke exposure. This can provide insights into the mechanisms underlying lung diseases associated with smoking, such as COPD. We find that repetitive CS exposure caused alveolar destruction as quantified by mean linear analysis (MLI) (Figure 6A, Figure 6B & Figure 6CD), (descriptive statistics can be found in Table S3). In addition to alveolar destruction, CS injury also increased cell death, as measured by lactate dehydrogenase (LDH) release assays (Figure 6D) (descriptive statistics can be found in Table S4). In addition, a mitochondrial membrane potential assay performed (TMRM) on air and CS-exposed human PCLS showed that slices exposed to CS had significantly lower mitochondrial membrane potential, indicating increased bioenergetic stress in the cells (Figure 6E), (descriptive statistics can be found in Table S5). CS injury also lowered the cellular metabolic activity in the tissue, as measured by Alamar Blue assays (Figure 6F), (descriptive statistics can be found in Table S6).

Figure 6.

Human precision-cut lung slices cigarette smoke exposure model. Three different normal human donors and seven replicate slices per experimental condition. P values are shown at the top of each graph in bold. (A) H&E of normal human lung exposed to humidified air. (B) H&E of normal human lungs exposed to cigarette smoke (CS). (C) LDH release of each replicate slice, shown as averages of triplicate values per slice. (D) MLI analysis of H&E slides shows the average width and height of vertical and horizontal chords measured per image. (E) TMRM fluorescence of human PCLS slices is shown as averages of triplicate values per slice. (F) Metabolic activity results obtained using the Alamar blue assay are shown as the average of the duplicate values of each slice. Scale bar 2 mm.

3.5. PCLS-A “mini lung” without a recruitable immune system

PCLS primarily consists of the structural components of the lung, including the epithelial cells, fibroblasts, and extracellular matrix, which are essential for maintaining the tissue architecture. However, immune cells play a critical role in the immune response and inflammation that occurs in the lungs when exposed to harmful agents like cigarette smoke. ^[30] PCLS lack the immune effector cells to mount a coordinated immunologic response to insults such as CS. To create a platform to study these responses, we isolated alveolar macrophages obtained from bronchoalveolar lavage of healthy mice. To track these macrophages, they were incubated with the cell-permeable dye, 5-chloromethylfluorescein diacetate (CFMDA) (Figure 7A). This dye is designed to freely pass through cell membranes into cells, where it is transformed into a cell-impermeant, fluorescent product, which is retained in living cells through several generations, allowing for cell-tracking. ^[25] CFMDA-labeled B6 alveolar macrophages were added to the surface of the PCLS to determine if they would incorporate into the tissue (Figure 7B, Figure 7C). PCLS with no added alveolar macrophages were used as controls and stained with Hematoxylin and Eosin (Figure 8A). We found alveolar macrophages incorporated into the alveolar walls and parenchyma (Figure 8B), suggesting their ability to migrate and integrate into the lung tissue similar to the migratory behavior of macrophages in the lung, which is required for immune surveillance and tissue homeostasis. Moreover, it indicated the ability of these added macrophages to potentially contribute to the immune response or tissue remodeling processes (Figure 7C). In fact, we found evidence of macrophage-epithelial crosstalk in the PCLS, as the presence of macrophages in the tissue caused a decrease polymerized actin was also observed in the epithelium in the mouse PCLS tissues as shown by live fluorescence confocal imaging using Cell Mask Actin tracking staining (Figure 7C). As increased actin is associated with epithelial injury, these studies show that the presence of macrophages is sufficient to induce a protective response in the tissue. ^[5]

Figure 7.

Fluorescence confocal live imaging of coculture murine PCLS. Images were taken with a 63x oil objective using the Zeiss LSM 880 Confocal with Airyscan FAST Module. Each experimental condition was performed in replicates of 6. Alveolar Macrophages (AMs) (green), F-actin (red), DAPI (blue). (A) CFMDA stained isolated alveolar macrophages. (B) control PCLS pre-stained with Cell Mask deep red Actin tracking stain plus added CFMDA labeled macrophages. (C) CS exposure experimental setup. (Air + AMs) PCLS with added alveolar macrophages exposed to air. (Second Air + AMs) different z-plane of PCLS exposed to air showing area with more infiltrating alveolar macrophages and less polymerized actin. (CS + No AMs) PCLS without added alveolar macrophages exposed to Cigarette smoke (CS). (CS + AMs) PCLS with added alveolar macrophages exposed to CS. (Second CS + AMs) different z-plane of PCLS exposed to CS showing area with more infiltrating alveolar macrophages and less polymerized actin. Scale bar: 10 μm.

Figure 8.

H&E histology Images of murine PCLS. Images were taken digitally using the Concentriq digital pathology website at 20x magnification. (A) H&E of PCLS with no added alveolar macrophages (AMs). (B) H&E of PCLS with added AMs (WT + Air-Airway) Airways of Wild Type (B6) PCLS exposed to air. (WT + Air-Alveoli) Alveoli of Wild Type (B6) PCLS exposed to air. (WT + CS-Airway) Airway of Wild Type (B6) PCLS exposed to cigarette smoke (CS). (WT + CS-Alveoli) Alveoli of Wild Type (B6) PCLS exposed to CS. Scale bar: 200 mm.

3.6. Considerations of a lung-on-a-chip model

We created a lung-on-a-chip to eliminate the membrane from the co-culture model while allowing for direct imaging of the cellular cross-section. Here, the main body of the chip is composed of three interconnected polydimethylsiloxane (PDMS) channels, with a smaller central channel in which a hydrogel solution was injected as a liquid and allowed to polymerize to create a barrier between the two outer channels (Figure 9A, Figure 9B). Human airway epithelial cells were seeded in a single cell suspension and inserted into one of the two side cellular channels, while the other compartment was filled with media. To create the co-culture, we mixed the hydrogel solution with THP-1 differentiated macrophage cells. Once the epithelial cells reached confluency, we removed the apical media to allow them to differentiate and form a barrier (Figure 9C). After transition to air-liquid interface, the epithelial cells formed a monolayer on the Matrigel hydrogel capable of keeping liquid from the media channel from filling the cellular channel for approximately 10-days, showing that the model established an effective epithelial barrier. The cells in the epithelial channel were only present on the vertical surface connected to the gel channel when transitioned to air-liquid interface, as that was the only surface capable of providing access to the media from the media channel. In the THP-1 co-cultured chips, THP-1 cells remained within the central hydrogel channel but also migrated to the cellular channel (Figure 9D). On a separate chip, HPMECs alone were seeded on a chip with empty Matrigel solution in the central channel. After testing diluted Matrigel, collagen solution, and attachment factor solution at various seeding densities, 500,000 cells were identified as the optimal seeding density to form a confluent endothelial monolayer on the channel surface (Figure 9E). The epithelial chip was analyzed using confocal microscopy and immunostaining of nuclei and E-cadherin (Figure 9F), which provided a visualization of the cellular interface. Although there was nonspecific signaling present in the gel matrix in the central channel of the chip, E-cadherin was present in cells identified with NucBlue nuclear stain. The endothelial monolayer was stained with NucBlue and anti-CD31 to confirm the presence of endothelial cells. (Figure 9G). Negative control images were taken for both sets of stains by taking images of the media channels (Figure S4.1, epithelial; Figure S4.2, endothelial) which received the staining protocol but were never seeded with cells.

Figure 9.

Culture of human airway epithelial cells on a lung-on-a-chip. (A) Schematic of the 3-channel lung chip showing the placement of epithelial cells and macrophages in each channel. The central chamber is filled with suspended macrophages in Matrigel. The epithelial cells grow on the interface between the epithelial and gel compartments at a 90-degree angle to form a barrier between the two compartments. The third channel provides media to the chip when the epithelial compartment is filled with air. (B) Image of the PDMS chip prior to seeding cells, scale bar: 3 mm. (C) Brightfield image of the interface between the epithelial and acellular gel compartment after epithelial cells have grown to confluence on the epithelial monoculture chip, scale bar: 300 μm. (D) Brightfield image of the interface between the epithelial and THP-1-seeded gel channel after epithelial cells have grown to confluence on the epithelial-immune coculture chip. Differentiated THP-1 macrophages are suspended in Matrigel in the gel channel, and have moved to the epithelial channel, scale bar: 300 μm. (E) Brightfield image of the endothelial chip, at the interface between the cellular channel and acellular gel compartment after human endothelial cells have grown to confluence, scale bar: 300 μm. (F) Confocal immunofluorescence image of the monoculture epithelial chip at air-liquid interface. Human bronchial epithelial cells stained for nuclei (blue) and E-cadherin (green) grow on the epithelial channel-gel compartment after 1 week of air-liquid interface culture, scale bar: 25 μm. (G) Confocal immunofluorescence image of endothelial chip in submerged culture. Human primary microvascular endothelial cells stained for nuclei (blue) and CD31 (red) grown in the endothelial channel, scale bar: 50 μm.

4. DISCUSSION

The lungs serve the vital role of allowing for oxygen exchange while at the same time maintaining a physical and immunologic barrier with the external environment. Different tissue structures and cell types support these functions, which are difficult to recapitulate in traditional in vitro culture models. Moreover, it has been challenging to study chronic lung diseases, which can take decades to develop in patients. ^[1] To address these limitations, we designed complementary microphysiological systems to model the airways and lung parenchyma, incorporating the in vivo crosstalk between different lung cell types. Furthermore, we were able to show that these models can exhibit relevant phenotypic changes to a pathologic stimulus, such as CS, to study the mechanics of COPD progression.

While airway epithelial cells require culture at an air-liquid interface to differentiate and maintain a functional barrier as a pseudostratified epithelium, monocytes are grown in submerged cultures and require specific treatments to differentiate into mature macrophages.^[31–32] In the lung, there is uncertainty about the origin and localization of macrophages within the alveoli and airways. Some persist within the lungs via self-renewal, while others differentiate from monocytes recruited to the lung.^[33] We have employed an epithelial-macrophage co-culture model that is able to sustain both a differentiated pseudostratified epithelial monolayer, and a population of alveolar macrophages and provides the milieu for alveolar macrophage-like expression. The presence of Siglec F on co-cultured PBMCs demonstrates that the epithelial microenvironment is sufficient to direct immune cell differentiation and function in the lung.^[14] Additionally, the induction of Siglec F expression on PBMCs supports the existing hypothesis that circulating monocytes are recruited to the lung to replace and/or supplement resident macrophage populations^{. [31–33]}

With prolonged CS exposure, the macrophages shifted from M2 anti-inflammatory markers to expressing M1 pro-inflammatory markers but can recover to the M2 phenotype. The M2 polarized macrophages seem to increase F-actin turnover in the epithelial cells and, thereby, are likely exerting protective effects on the epithelial monolayer in both the mouse and human models. With CS injury, the actin dynamics are dominated by the stable, longer-lived actin stress fibers in the injured and diseased cells, ^[5] which is not altered by the presence of macrophages, potentially reflecting the crosstalk between M1 pro-inflammatory macrophages and the epithelial cells. Further studies are needed to dissect the mechanisms mediating this crosstalk to see if it could be harnessed for therapeutic intervention.

While human airway epithelial cells on an air-liquid interface (ALI) continue to be invaluable, studying alveolar damage is more complicated as these models lack the complex three-dimensional architecture and intricate multicellular interactions found in the alveolar compartment.^[34–36] Additionally, animal models of COPD, which are traditionally used to study alveolar destruction can take several months to develop and inflict high stress levels on the animals while only partly recapitulating human disease ^[33]. The use of living three-dimensional lung tissues as precision-cut lung slices (PCLS) allows for the investigation of disease in structurally relevant regions of the lungs, where all native cell types, extracellular matrix, and architecture are present and is a promising and relevant human model to study respiratory responses to inflammatory stimuli and infections and test pharmaceutical compounds’ safety. ^[36] In this study, we used precision-cut lung slices derived from non-transplantable human lungs to optimize a CS exposure model. We found that exposure of human precision-cut lung slices to eight cigarettes was enough to cause alveolar destruction, increased cytotoxicity, decreased metabolic activity, and decreased mitochondria integrity. Our findings show that exposing human-derived precision-cut lung slices to CS can model human alveolar destruction in a few days and provides a structurally and physiologically relevant model to understand the molecular mechanisms of COPD progression and heterogeneity.

Although precision-cut lung slices provide the structural environment to study the effects of CS and respiratory diseases like COPD, they still lack immune effector cells. To better understand the cell-cell interactions between resident lung cells and infiltrating immune cells when exposed to CS, we co-cultured mouse PCLS with alveolar macrophages derived from the same mouse.

Our data showed that alveolar macrophages could penetrate the alveolar walls and migrate to the airway epithelium. These findings represent a significant advance in using PCLS to study disease mechanisms, as different immune effector cells can be introduced into the PCLS to characterize the phenotypic immune cell profile observed in respiratory diseases like COPD.

We found that the alveolar epithelial-macrophage interactions in the PCLS mirrored the airway epithelial-macrophage effects. The PCLS co-cultured with alveolar macrophages and exposed to short CS exposures showed decreased F-actin and reduced CS-induced stress fiber formation compared to the PCLS without alveolar macrophages. This observation corroborates our short CS exposures in the co-culture experiments, where the presence of M2 macrophages improved actin turnover in the epithelium to reduce stress fiber formation. Future studies can determine the CS exposure required to convert the macrophages in PCLS to M1 pro-inflammatory cells to see if this model has similar reductions in actin turnover. ^[37] However, these correlations between MPS models highlight the power of parallel analysis of different in vitro models in their ability to provide context for the mechanisms of cell behaviors and diseases.

Sophisticated model platforms are needed to study complex cellular crosstalk in modulating molecular responses to stimuli.^[37–38] The PCLS plus added immune cells system provides a more physiologically relevant model to study not only inflammation and the safety of pharmaceutical compounds but also the different immune cell subtypes and their interactions with the local tissue environment.^[36] In addition to biological advantages, PCLS provides a more ethical alternative to studying disease by reducing the number of animals used for an experiment and decreasing the amount of experiment-induced stress that animals experience in traditional smoking chambers.

The lung-on-a-chip model provides an adaptable system that allows for the live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways, as well as detailed cellular mechanistic dissection. The organ-on-a-chip system can incorporate biologically relevant architecture and physical and biochemical cues to mimic the in vivo environment. Furthermore, the use of an ECM substrate and PDMS chip body provides a softer growth surface similar to soft tissue ^[39], as opposed to the rigid polystyrene membrane of a Transwell. Substrate stiffness can have significant effects on cell behavior, and growing the epithelial cells on either the PDMS body of the chip or the surface of the ECM gel without the presence of a rigid pre-formed membrane provides a more physiologically relevant mechanical environment. In this model, the epithelia can maintain a barrier, show evidence of differentiation, and allow cross-section imaging. Unlike the Transwell model, which requires slicing cross-sections post-fixation, the chip platform can be imaged at the cross-section. Cross-sectional imaging provides a better evaluation of the co-culture and a detailed analysis of epithelial plasticity with injury. Additional applications could include patient-derived fibroblasts embedded in the central hydrogel channel to study pathologies that involve pulmonary fibrosis ^[40]. It can be further adapted to model alveolar epithelial interactions as it can sustain the inclusion of endothelial cells, providing the milieu required to model the lung alveoli. This configuration would allow for the analysis of movement of monocytes across the endothelium, through the gel channel where there is no intervening membrane, and to the epithelial channel to model the movement of PBMCs to the airway lumen in response to insults. Beyond these advantages for more biologically relevant models, they can also include design elements that make them ideal for examining cells with precise laboratory techniques, such as high-resolution microscopy.

Of course, these are all models of chronic disease and are not fully representative of the complexities of human disease that occurs over decades, such as COPD. However, as we cannot dissect molecular mechanisms over that prolonged time frame in human patients, and animal models also do not fully capture all the clinical aspects of COPD pathology. Each MPS model evaluated in this study offers unique advantages to study aspects of pulmonary physiology in the context of respiratory disease. Using a combination of these models provides a complementary approach to better address the intricate mechanisms of lung disease.