Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

Summary

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

Abstract

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

Introduction

The human lung has a remarkable role in respiration and immune defense, with complex interactions between the immune responses of the alveolar mucosa¹. The ability of the alveoli to create an efficient immune response is vital for preventing lung infections and securing pulmonary health. Since the lungs are constantly exposed to a wide range of potential risks, including bacteria, viruses, fungi, allergies, and particulate matter, understanding the complexities of alveolar mucosal immune responses is critical for discovering the mechanisms behind respiratory infections, inflammatory disorders and treating pulmonary diseases¹.

To study infection and inflammation-related processes of the respiratory tract in vitro, models that could faithfully mimic the alveolar milieu and the immune responses are required. 2D cell culture and animal modules have been used for decades as essential tools for biomedical research on lung immune response. However, they often have limitations in their translational potential to human situations. Lung-on-chip models can contribute to filling the gap between traditional in vitro and in vivo models and provide a novel approach to studying human-specific immune responses^2,3. Lung-on-chip models can mimic the air-liquid interface, which is required for lung cells to recapitulate physiological conditions of the respiratory tract and to develop a more accurate and robust tissue model. This culture technique enables a precise examination of cell differentiation, functioning, and responses to drugs or disease-related stimuli in vitro².

In this study, we present a microfluidic-based model of the human alveolus as an effective tool to recapitulate the human alveolar milieu by applying perfusion to mimic blood flow and biomechanical stimulation of endothelial cells and incorporating an air-liquid interface with epithelial cells exposed towards an air phase⁴. We have developed a microfluidic perfused alveolus-on-chip that mimics the physical structure and biological interactions of the human alveolus, with a particular focus on the air-liquid interface. This interface plays a crucial role in the differentiation of respiratory epithelial cells, which is essential for accurately modeling the pulmonary environment. The model uses human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs), separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages positioned between the cell layers. This setup replicates the intricate cellular arrangement of the alveolus and is critical for accurately simulating the air-liquid interface, which is a significant factor in the physiological function of lung tissue.

The rationale behind the model development extends to integrating both circulating and tissue-resident immune cells. This approach is designed to accurately mimic the inflammatory host response to human respiratory infections, providing a dynamic environment to study pathogen-host interactions. The presence of macrophages allows for the examination of immediate immune responses and their interaction with pathogens, reflecting the first line of defense against respiratory infections. Furthermore, the design of the biochip platform facilitates convenient and precise manipulation of both biophysical and biochemical cues, which is crucial for replicating alveolus function in vitro. This flexibility is instrumental in dissecting the contributing factors to human infections, enabling researchers to adjust conditions to reflect various disease states or to test potential therapeutic interventions. The compatibility of the platform with multiple readout technologies, including advanced microscopy, microbiological analyses, and biochemical effluent analysis, enhances its utility. These capabilities allow for a comprehensive assessment of the tissue response to infections, including evaluating cellular behavior, pathogen proliferation, and the effectiveness of immune responses.

We present a detailed protocol and techniques to create and utilize a human alveolus-on-chip model focused on replicating the air-liquid interface and integrating immune cells to study human infections in vitro.

Protocol

HUVEC cells are isolated from umbilical cords and used up to passage 4. Primary monocytes are isolated from healthy donors from whole blood. The study was approved by the ethics committee of the Jena University Hospital, Jena, Germany (3939-12/13). According to the Declaration of Helsinki, all individuals donating cells for the study gave their informed consent.

1. Day 1: Preparation of the biochip

1. The biochips are available in different sizes and models. For experiments here, use the model BC002. Place the biochip in a glass Petri dish, fill the dish, and flush all cavities with sterile 70% ethanol (EthOH). Incubate for 45 min at room temperature. Afterwards, flush cavities 2x with sterile ddH₂O. Remove the ddH₂O.
2. Coat the biochip membrane of both cavities with sterile collagen IV solution (0.5 mg/mL in 0.5 mM acetic acid; use 1:100 concentration in Dulbecco's phosphate buffered saline containing magnesium and calcium PBS (+/+)) by gently flushing the collagen IV solution through both cavities using a 1,000 µL pipet with blue tips. Incubate for 15 min at 37 °C and 5% CO₂.
   NOTE: Using a 1,000 µL pipet with blue filter (P1000) tips is recommended throughout the entire protocol. The diameter of the opening of the filter tip completely covers the opening of the ports connecting the channels, which avoids the insertion of air bubbles into the chip.
3. After 30 min incubation, flush the lower and upper chambers 2x with sterile PBS (+/+) to flush out the remaining collagen and acetic acid.
4. Prepare EC medium by adding 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL). Fill the upper chamber of each cavity with 250 µL of EC medium and the lower chamber with 150 µL of EC medium.
5. Harvest HUVEC from a confluent T25 flask: Wash with 5 mL of Dulbecco's phosphate buffered saline without magnesium and calcium PBS (-/-), add 1 mL of 0.25% Trypsin + 1 mM EDTA in PBS (-/-) and incubate for 3 min at 37 °C. Stop trypsin activity by adding 5 mL of PBS (+/+) + 5% FCS and centrifuge at 350 x g for 5 min at room temperature (RT) in a 50 mL conical tube. Remove the supernatant and resuspend the cell pellet in 1 mL of EC medium.
6. Use a 1:10 serial dilution when performing cell counting. Add 10 µL of the resuspended cell pellet to 90 µL of cell medium. Combine the samples carefully. Use a microcentrifuge tube with 10 µL of trypan blue stain. Dilute the cell solution with trypan blue (1:1) using cell counting chamber slides. Count the cells either manually with a Neubauer Chamber or with an automated cell counter. Determine cell counts and adjust concentration to 0.4 x 10⁶ cells per 200 µL.
7. Replace the endothelial cell medium in the upper chamber of the biochip with 200 µL of EC medium containing suspended HUVECs. Pipette gently to avoid the formation of air bubbles in the channels or chambers. Place the glass Petri dish with the biochips at 37 °C and 5% CO_2.

2. Day 2: Checking of the cell layer

1. On Day 2, visually check for the confluency of the HUVEC cell layer in the upper chamber. Perform medium exchange in the upper chamber with 300 µL of EC medium. Pipette gently to avoid detachment of cells from the membrane. Perform media exchange daily after 24h. Place the glass Petri dish with the biochips back at 37 °C and 5% CO₂.

3. Day 3: Preparation of lower chamber

1. Visually check the confluency of the endothelial cells layer. At this point, determine the presence of a confluent layer and cobblestone-like morphology (Figure 1). Perform medium exchange in the upper chamber with 300 µL of EC medium.
2. Next, prepare the lower chamber for seeding the cells forming the epithelial layer. Flush the lower chamber gently with 150 µL of the RPMI medium (add 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 5% fetal cow serum, 1 µM dexamethasone).
3. For the lower chamber, seed 0.5 x 10⁶ H441 cells suspended in 200 µL of RPMI medium (RPMI) per cavity.
4. Harvesting H441 cells
   1. Wash the cells with 5 mL of PBS (-/-) once. Add 2 mL of 0.25% Trypsin + 1 mM EDTA in PBS (-/-).
   2. Incubate the cells for 5 min at 37 °C and 5% CO₂. The incubation time varies depending on the confluency of the layer. Within 5-7 min, cells should detach completely.
   3. Stop the trypsin activity with 10 mL of PBS (+/+) + 5% FBS, collect the cells, and centrifuge at 200 x g for 4 min.
   4. After centrifugation, discard the supernatant, resuspend the pellet in 1 mL of RPMI, and count.
   5. Seed 0.5 x 10⁶ cells per chamber by gently pipetting the cell solution into the lower chamber. Close all the ports and flip the biochip upside-down so the cells can attach to the PET membrane. Keep the biochip upside-down and incubate overnight in a cell culture incubator at 37 °C and 5% CO₂.

4. Days 4 - Day7: Maintenance of cells and harvesting monocytes

1. Perform daily medium exchange in the upper and lower chamber. For the upper chamber, perform the medium exchange with 300 µL of EC medium.
2. Use 200 µL of RPMI+ (RPMI medium plus 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 10 ng/mL GM-CSF, 10% autologous human serum, 1 µM dexamethasone) for the lower chamber. On day 7, flush the lower chamber with RPMI+ to prepare the cavity for seeding monocytes.
3. Seed 0.1 x 10⁶ monocytes suspended in 200 µL RPMI+ in the lower chamber.
4. Harvesting monocytes
   1. Wash the cells with 1 mL of PBS (-/-) and incubate for 7 min at 37 °C in pre-warmed PBS (-/-) containing 4 mg/mL Lidocaine + 5 mM EDTA at 37 °C and 5% CO₂.
   2. Collect the cells and centrifuge at 350 x g for 7 min, resuspend the cells in 1mL of RPMI+, and count. Place the biochip ports facing downwards so the cells attach to the membrane.

5. Day 8: Biochip perfusion

1. At this point, ensure the biochips are ready to be connected to the flow and perfused. Before starting the perfusion, prepare an empty incubator and place the peristaltic pump inside.
2. Perfusion of the biochips
   1. Before attaching the sterilized tubing to the biochip, flush the tubing with 200 µL of PBS (+/+), followed by 200 µL of EC medium. The tubing has an inner diameter of 0.5 mm, consisting of longer (20 cm) and shorter (12 cm) sides divided by two peristaltic pump stoppers. Put the tubing aside and prepare the reservoirs for the medium.
   2. Perform medium exchange in the upper and lower chambers by flushing them with the respective freshly prepared cell culture medium.
   3. Insert the reservoir on the outlet side of the biochip and connect the reservoir with the tubing to the inlet of the biochip (Figure 2). Connect the tubing to the reservoir and the biochip so that there is a circular medium perfusion between the reservoir and the biochip.
   4. Once everything is connected to the ports, fill in the reservoir with a 500 µL of EC medium. Connect the biochip with the tubing on a perfusion with a flow rate of 21 µL/min (Figure 2). Observe closely during the first 15 min of perfusion for the insertion of bubbles. Perform medium exchange daily in both chambers.

6. Day 9 - Day 11: Establishing an air-liquid interface

1. Stop the perfusion, empty the reservoirs, and refill with freshly prepared medium under a sterile safety cabinet. Pipette 500 µL of EC medium into the reservoirs and flush gently the lower chamber with 200 µL of RPMI+. Start the perfusion again with a perfusion rate of 21 µL/min.
2. Air Liquid Interface (ALI)
   1. Establish ALI from day 12 until the end of the experiment (day 14). During ALI culture, expose the epithelial cells to air.
   2. To establish the ALI culture, prepare a new vascular medium EC (add 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 10 ng/ml GM-CSF, 20% autologous human serum, 1µM dexamethasone) to maintain all cell types in the chip. At this point, the medium contains a higher concentration of AS (20%).
   3. Open the plugs at the lower chamber under a sterile safety cabinet and carefully soak up the medium until an air bubble is visible, as shown in Figure 3. Ensure the entire liquid from the channel and the chamber is removed and close the ports carefully.
   4. Fill in the reservoirs with EC (-/-) medium and continue perfusion culture with a flow rate of 21 µL/min. Exchange medium only at the upper chamber until day 14 and keep the epithelial cells in the lower chamber with air only.

7. Day 14: Tissue harvesting and analysis

1. Disconnect the biochip from the flow. Plug out the tubing and check on the microscope for cell confluency. Due to the air in the lower chamber, the cell layers might appear fuzzy and difficult to visualize. At this point, the model is ready for further experimentation.
2. Fixation and staining of tissue in biochip
   1. Wash the cavities with PBS (+/+) to remove the cell culture medium. Pipette 300 µL of 4% paraformaldehyde (PFA) dissolved in PBS (-/-) to the upper chamber and 200 µL in the lower chamber and incubate for 10 min at RT.
      NOTE: When working with fixation chemicals such as PFA, use a fume hood. Collect and dispose of the waste accordingly.
   2. Wash the chambers 3x with PBS (+/+). After fixation, store the biochips at 4 °C or use them directly for immunofluorescence staining of cells.
   3. For permeabilization, pipette 300 µL of 0.25% Triton X-100 in PBS (+/+). Incubate for 30 min at RT.
   4. After incubation, wash both chambers with PBS (+/+) and pipette 300 µL of 3% Bovine serum albumin (BSA) in PBS (+/+) in both chambers. Incubate for 1 h at RT.
   5. In the meantime, prepare the antibody panel for immunofluorescence staining. To prepare the antibody panel for immunofluorescence staining, a tube with a sufficient amount of 3% BSA was set up to dilute the antibodies. Use 50 µL of antibody dilutions for each membrane. Use a dilution ratio of 1:100 for primary antibody panels and 1:200 for secondary antibody panels. Additionally, for the secondary antibody panel, prepare DAPI at a dilution ratio of 1:2,000. For corresponding antibodies, see (Table 1).
   6. To directly access the cells within the biochip, the bonding foil must be removed. To open the microfluidic chambers, make an exact cut across the outside of the cavity and remove the bonding foil. Cut the membrane out by removing the edges sealed to the biochip with the scalpel.
      NOTE: Work precisely and accurately during this step to avoid any membrane damage and injuries due to the sharpness of the scalpel.
   7. After detaching the membrane from the biochip, divide the membrane into two pieces. Collect the membrane pieces with a tweezer and place them on a microscopic slide for staining.
   8. Pipette 50 µL of antibody solution per membrane piece. Incubate overnight at 4 ˚C, protected from light.
   9. After incubation, place the membrane in 500 µL of PBS (-/-) in a 24 well-plate and wash the membrane 3x for 5 min each in PBS (+/+). After washing, pipette 50 µL of secondary antibody solution and incubate for 1 h at RT protected from light.
   10. Wash the membrane with PBS (+/+) 3x for 5 min each. Meanwhile, prepare and label the microscopic slides for the experiment. Place a drop of mounting medium on the slide and place the corresponding membrane. Repeat this step for all membranes. At the end, place one more drop of the mounting medium on the top of the membrane and use a cover glass. Store at 4 ˚C.

Results

An examination of morphological alterations and the expression of marker proteins could be performed using immunofluorescence staining. After co-culturing for 14 days, the vascular and epithelial sides are analyzed for expression of respective cell markers. This method is useful for studying the interactions and integrity of vascular and epithelial components, which is essential for disease modeling as a functional biological readout related to infection. Immunofluorescence staining could be supported by quantifying anal...

Discussion

The alveolus-on-chip model represents a multilayered tissue model of the human alveolus, integrating essential cell types of the lower respiratory tract, including lung epithelial cells, endothelial cells, and macrophages, cultured in an organotypic arrangement at an ALI with medium perfusion of the endothelial lining. Cells of different layers express specific cell marker proteins such as E-cadherin, a calcium-dependent adhesion molecule of lung epithelial cells, which is central in establishing intercellular epithelial...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Consumables
Cellcounting chamber slides (Countess)	Invitrogen	C10283
Cell culture Multiwell Plates, 24 Well, steril	Greiner Bio-One	662 160
Cell culture Multiwell Plates, 6 Well, steril	Greiner Bio-One	657 160
Coverslips (24x40mm; #1.5)	Menzel-Gläser	15747592
Eco wipes	Dr. Schuhmacher	00-915-REW10003-01
Eppies 2.0	Sarstedt	72.691
Eppis 0.5	Sarstedt	72.699
Eppis 1.5	Sarstedt	72.690.001
Falcons 15mL	Greiner Bio-One	188 271-TRI
Falcons 50mL	Greiner Bio-One	227 261-TRI
Gauze swab	Noba	PZN 2417767
Gloves Nitril 3000	Meditrade	1280
Microscope slides	Menzel-Gläser	AAAA000001##12E
Multiwell Plates 24 Well, sterile	Greiner Bio-One	662 160
Pasteur pipettes (glass) 150mm	Assistent	40567001
Pasteur pipettes (glass) 230mm	Assistent	40567002
Round-bottom tubes (PS, 5mL)	Falcon	352052
Safety-Multifly-Set, 20G, 200mm	Sarstedt	85.1637.235
Scalpels	Dahlhausen	11.000.00.715
Serological pipettes 10mL	Greiner Bio-One	607 160-TRI
Serological pipettes 25mL	Greiner Bio-One	760 160-TRI
Serological pipettes 2mL	Greiner Bio-One	710 160-TRI
Serological pipettes 50mL	Greiner Bio-One	768 160-TRI
Serological pipettes 5mL	Greiner Bio-One	606 160-TRI
S-Monovette, 7,5ml Z-Gel	Sarstedt	1.1602
S-Monovette, 9,0ml K3E	Sarstedt	02.1066.001
Softasept N	Braun	3887138
T25 flask	Greiner Bio-One	690 960
Tips sterile 10µL	Greiner Bio-One	771 261
Tips sterile 1250µL	Greiner Bio-One	750 261
Tips sterile 300µL	Greiner Bio-One	738 261
Tips unsterile 10µL	Greiner Bio-One	765 290
Tips unsterile 1000µL	Greiner Bio-One	739 291
Tips unsterile 200µL	Greiner Bio-One	686 290
Tweezers (Präzisionspinzette DUMONT abgewinkelt Inox08, 5/45, 0,06 mm)	Roth	K343.1
Chemicals
Descosept AF	Dr. Schuhmacher	N-20338
Ethanol 96%	Nordbrand-Nordhausen	410
Fluorescein isothiocyanate (FITC)-dextran (3-5kDa)	Sigma Aldrich	FD4-100MG
Fluorescent Mounting Medium	Dako	S3023
Methanol	VWR	20847.295
Saponin	Fluka	47036
Tergazyme	Alconox	1304-1
Cell culture
Collagen IV	Sigma-Aldrich	C5533-5MG
Dexametason	Sigma-Aldrich	D4902
DPBS (-/-)	Lonza	BE17-516F
DPBS (+/+)	Lonza	BE17-513F
EDTA solution	Sigma-Aldrich	E788S
Endothelial Cell Growth Medium	Promocell	C-22020
Endothelial Cell Growth Medium supplement mix	Promocell	C-39225
Fetal bovine Serum	Sigma-Aldrich	E2129-10g
H441	ATCC
Human recombinant GM-CSF	Peprotech	300-30
Lidocain	Sigma-Aldrich	L5647-15G
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140-122 /-163
RPMI	Gibco	72400047
Trypane blue stain 0.4%	Invitrogen	T10282
Trypsin	Gibco	15090-046
Primary antibodies
Cadherin-5 / VE-Cadherin (goat)	BD	610252
CD68 (rabbit)	CellSignaling	76437
E-Cadherin (goat)	R&D	AF748
SP-A (mouse)	Abcam	ab51891
Secondary antibodies
AF488 (donkey anti mouse)	Invitrogen	R37114
AF647 (donkey anti mouse)	invitrogen	A31571
AF647 (donkey anti rabbit)	Invitrogen	A31573
Cy3 (donkey anti goat)	jackson research	705-165-147
Microfluidic
Chip	Dynamic 42	BC002
Male Luer Lock (small)	ChipShop	09-0503-0270-09
Male mini luer plugs, row of four,PP, green	Microfluidic chipshop	09-0558-0336-11
Male mini luer plugs, row of four,PP, opaque	Microfluidic chipshop	09-0556-0336-09
Male mini luer plugs, row of four,PP, red	Microfluidic chipshop	09-0557-0336-10
Plugs	Cole Parmer	GZ-45555-56
Reservoir 4.5mL	ChipShop	16-0613-0233-09
Tubing	Dynamic 42	ST001
Equipment
Autoclave	Tuttnauer	5075 ELV
Centrifuge	Eppendorf	5424
CO2 Incubator	Heracell	150i
Countess automated cell counter	Invitrogen	C10227
Flowcytometer	BD	FACS Canto II
Freezer (-20 °C)	Liebherr	LCexv 4010
Freezer (-80 °C)	Heraeus	Herafreeze HFU 686
Fridge	Liebherr	LCexv 4010
Heraeus Multifuge	Thermo Scientific	X3R
Microscope	Leica	DM IL LED
Orbital shaker	Heidolph	Reax2000
Peristaltic pump	REGLO Digital MS-4/12	ISM597D
Pipettes 10µL	Eppendorf Research plus	3123000020
Pipettes 100µL	Eppendorf Research plus	3123000047
Pipettes 1000µL	Eppendorf Research plus	3123000063
Pipettes 2.5µL	Eppendorf Research plus	3123000012
Pipettes 20µL	Eppendorf Research plus	3123000039
Pipettes 200µL	Eppendorf Research plus	3123000055
Scale	Sartorius	6101
Scale	Sartorius	TE1245
Sterile bench	Kojair	Biowizard SL-130
Waterbath	Julabo	SW-20C
Fluorescence Microscope Setup
Apotome.2	Zeiss
Illumination device	Zeiss	HXP 120 C
Microscope	Zeiss	Axio Observer 5
Optical Sectioning	Zeiss	ApoTome
Power Supply Microscope	Zeiss	Eplax Vp232
Software
ZEN Blue Edition	Zeiss