Generation of 3D Collagen-Embedded A549/MRC-5 Alveolar Organoids using a Mechanical Cell-Stretching Bioreactor

Summary

This protocol outlines the generation of organoids from A549 alveolar epithelial cells and MRC-5 lung fibroblasts on an extracellular matrix hydrogel scaffold, followed by embedding in 3D collagen gels and exposure to mechanical strain using a cell-stretching bioreactor to investigate alveolar mechanobiology.

Abstract

Although breathing dynamics and mechanical strain play critical roles in alveolar biology, their contributions to the pathogenesis of respiratory diseases are often overlooked. To address this gap, we developed an organoid model in which alveolar epithelial-fibroblast organoids are subjected to controlled mechanical strain using a cell-stretching bioreactor. This platform replicates the spatial organization and multicellular complexity of alveolar tissue and enables investigation of how cell-cell communication and mechanotransduction influence tissue structure and function in health and disease. In this protocol, we describe methods for generating and culturing epithelial-fibroblast organoids using A549 alveolar epithelial and MRC-5 lung fibroblast cell lines in a basement membrane extracellular matrix hydrogel. Organoids are then non-enzymatically isolated and embedded in 3D collagen gels in specialized plates in the bioreactor. We outline procedures for applying equibiaxial strain to mimic pathological breathing patterns seen in respiratory exacerbations. Finally, we present a standard characterization workflow, including immunostaining for cell-specific markers and immunoassays to profile immune mediator release. Together, these analyses support the assessment of organoid structure and function. This protocol provides a user-friendly, reproducible, and adaptable platform for studying alveolar biology under mechanical stress, with easily modifiable parameters to meet diverse experimental goals and advance research into lung disease mechanisms.

Introduction

The alveoli are the terminal sacs of the respiratory system and serve as the primary site of gas exchange. They are composed mainly of alveolar type I and II epithelial cells, responsible for gas exchange, immune functions, and maintenance of structural integrity, and fibroblasts within the walls, which maintain extracellular matrix homeostasis¹. These and other resident cells are continuously exposed to cyclic stretch and shear stress during respiration². Such mechanical cues are transduced into biochemical signals that influence cell structure, differentiation, and communication³.

Pathological mechanical strain is observed in lung diseases such as chronic obstructive pulmonary disease (COPD). COPD is a progressive condition characterized by parenchymal destruction, chronic inflammation, and narrowing of the small airways⁴. In this disease, destruction of alveolar walls, loss of elastin, extracellular matrix (ECM) remodeling leading to fibrosis, and chronic inflammation collectively impair the lung's ability to withstand structural damage^5,6. This results in heterogeneous and abnormal alveolar deformation during respiration, which is further exacerbated during disease flare-ups^7,8.

While several in vitro approaches have been used to study alveolar biology, each has notable limitations. 2D co-culture systems facilitate the investigation of cell-cell interactions but fail to capture tissue architecture or extracellular matrix dynamics, reducing physiological relevance. Static 3D cultures better preserve structural organization and matrix interactions, yet they do not incorporate dynamic mechanical cues present in vivo. Microfluidic and organ-on-chip platforms can mimic fluid flow and mechanical strain, providing high physiological fidelity, but they are often technically complex, low-throughput, and costly⁹. In contrast, the 3D mechanically strained organoid model presented here combines structural complexity, cellular heterogeneity, and controlled mechanical stimulation, an aspect often overlooked in existing models, within a reproducible, scalable platform that offers a balance of physiological relevance and experimental accessibility^10,11.

To understand the complex mechanisms that contribute to diseases such as COPD and discover therapeutic targets, disease models have been established. Despite its importance, mechanical strain is often overlooked in the in vitro alveolar models used to study the mechanisms and pathogenesis of lung diseases such as COPD. To address this gap, we present a method for culturing alveolar epithelial-fibroblast organoids in a basement matrix, retrieving and embedding them in 3D collagen gels, applying cyclical mechanical strain using a cell-stretching bioreactor, and evaluating key structural and functional outcomes. These 3D mechanical organoid models better capture cellular interactions and the microenvironment of the human alveolar niche, enabling the study of epithelial-mesenchymal crosstalk¹².

The bioreactor applies equibiaxial strain with configurable parameters, allowing up to 30% elongation and frequencies of up to 5 Hz to model both physiological and pathological conditions. In this study, we generated organoid models embedded in collagen-I hydrogels subjected to 18% strain at 0.4 Hz for 24 h to mimic pathological breathing. We established a standardized characterization workflow that includes immunostaining for cell-specific markers and immunoassays to quantify immune mediator release following mechanical strain. The goal of this study is to provide a physiologically relevant, user-friendly, and adaptable platform for investigating alveolar biology under mechanical stress.

Protocol

All steps require aseptic technique, sterile materials, and reagents, and are performed in a biosafety cabinet to reduce exposure of personnel to biohazardous material and contamination of cultures. The reagents and the equipment used are listed in the Table of Materials.

1. Cell ​culture and establishment of cell stocks

1. Prior to organoid formation, culture A549 and MRC-5 cells on standard tissue culture-treated plastic flasks (T75 cm²) to approximately 70%-80% confluency using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, hereby referred to as "complete DMEM".
2. For both A549 and MRC-5, seed at 2 x 10⁴ cells/cm², in a total volume of 10 mL complete DMEM. Culture cells at 37 °C with 5% CO₂ in a humidified environment until approximately 70%-80% confluency is reached.
3. Replace the culture medium every 48 h to ensure optimal growth conditions.
4. Subculture by aspirating all media from the flask once 70%-80% confluent, wash once with warm phosphate-buffered saline (PBS), then add 4 mL of trypsin-EDTA.
5. Incubate at 37 °C for 5 min and visualize the flasks with a brightfield microscope to ensure complete detachment. Neutralize by adding 4 mL of complete DMEM.
6. Count the cells using the technique of choice, such as trypan blue exclusion counting.
7. Centrifuge the cell suspension at 200 x g for 5 min.
8. Aspirate the supernatant to leave a cell pellet at the bottom of the conical tube. Resuspend the cell pellet in an appropriate volume of complete DMEM by triturating gently.
9. For freezing, resuspend in complete media containing 10% DMSO to yield a 1 x 10⁶ cells/mL. For organoid formation, resuspend to get 1 x 10⁶ cells/mL.

2. Establishing and characterizing the maturation of a 3D alveolar epithelial-fibroblast organoid model

NOTE: The basement matrix should be thawed on ice overnight and kept on ice during these steps. Higher temperatures can cause solidification of the matrix.

1. Prepare a 2:3 mixture of basement matrix and advanced DMEM. Add 300 µL of this solution to each well of a 24-well plate, ensuring the bottom of each well is evenly coated. Incubate the plate at 37 °C in a humidified incubator for 1 h to allow the matrix to solidify.
2. Prepare a 5% basement matrix solution in complete DMEM, which will be the culture medium for the organoids. For each well, make 1.2 mL of this solution by diluting 60 µL of basement matrix in 1,140 µL of complete DMEM.
3. During the time that the basement matrix mixture is solidifying, mix 300,000 A549 cells with 30,000 MRC-5 cells in 200 µL of the 5% basement matrix solution made in complete DMEM described in Step 2.2.
4. Once the 2:3 basement mixture has solidified, pipette 200 µL of the A549/MRC-5 mixture onto the gelled matrix, then return the 24-well plate to incubate at 37 °C, 5% CO₂ for 21 days to allow for growth and differentiation of the alveolar organoids.
5. Visualize the cultures under a light microscope for the next 48 h. Cells should be aggregating within this time period.
6. Replace the 5% basement matrix/DMEM solution every 48 h with 200 µL of fresh 5% medium. To avoid disturbing the organoids embedded in the hydrogel, gently tilt the plate so the old solution collects along the well wall before aspiration.
7. At days 3, 9, and 21 of maturation, image the organoids at 10x objective magnification and take four representative images or fields of view of each well. Representative images of early organoid morphology at days 3, 9, and 21 are shown in Figure 1.
8. For each field of view, count all organoids larger than 50 µm in diameter. Using the known area of the field of view, extrapolate the average total number of organoids per well from the average count per field, assuming a 24-well plate surface area of 1.9 cm².
   NOTE: These analyses can be done using image analysis software such as Fiji¹³.
9. Average diameter and area of the organoids can also be measured to obtain quantitative information about the growth characteristics of the organoids in culture.

3. Harvesting 3D alveolar epithelial-fibroblast organoids

NOTE: All steps to harvest the organoids should be performed on ice.

1. After 21 days of maturation, aspirate the supernatant and wash gently with approximately 1-2 mL of ice-cold PBS.
2. Add 1-2 mL of cold organoid harvesting solution and incubate the plate at 2-8 °C for 30-90 min with moderate shaking.
   NOTE: Stop the incubation when the organoids are seen floating at the bottom of the well.
3. Monitor the dissociation using brightfield microscopy. Dissociation is considered complete once the basement matrix at the bottom of the well is no longer visible and the organoids are in suspension. A cell scraper can be used to gently dislodge the organoid-basement matrix hydrogel to accelerate the process.
4. Pool the contents of all wells into a 50 mL conical tube on ice.
5. Centrifuge at 500 × g for 5 min at 2-8 °C. Aspirate the supernatant, wash the organoid pellet with 1-2 mL of ice-cold PBS, then centrifuge again to obtain a pellet. Aspirate the PBS.

4. Establishing 3D collagen-embedded alveolar-fibroblast organoids and mechanical strain application

NOTE: Use circular foam culture plates with foam anchors compatible with a cell-stretching bioreactor. The culture plate used in this protocol has a growth area of ~6.6 cm².

1. Prepare a working solution of 2 mg/mL collagen I gel. The solution should contain 1× PBS, 11.4 mM NaOH, and 2 mg/mL collagen I, with the final volume adjusted using sterile water. Prepare 2 mL of this collagen I solution per well of the circular foam 6-well plate.
2. Add 1 mL of the 2 mg/mL collagen I solution to the foam anchors on the periphery of the plate, ensuring complete saturation.
   NOTE: The foam anchors must be thoroughly saturated with collagen I solution to ensure proper adhesion. Incomplete saturation can result in detachment of the collagen gel during mechanical strain application, leading to a reduced effective strain compared to the intended target.
3. Resuspend the organoid pellet in 6 mL of 2 mg/mL of rat tail collagen and pipette 1 mL of the suspension into the middle of a 6-well circular foam plate, ensuring the organoid-infused collagen mixture mixes with the initial organoid-free collagen added to the circular foam anchors.
   NOTE: Avoid generating bubbles when adding the organoid suspension into each well.
4. Polymerize the collagen-I for 1 h by leaving the plates in an incubator (37 °C, 5% CO₂)_. After polymerization, the gel should appear cloudy.
5. Add 2 mL of complete DMEM on top of 3D-collagen-embedded A549/MRC5 organoids and return the plate to the incubator for 24 h.
6. Replace the culture medium with DMEM + 1% FBS and incubate overnight prior to mechanical strain exposure.

5. Cell stretching bioreactor setup and exposure to mechanical strain

1. Insert loading stations into each well of the baseplate. These contain six 25 mm diameter loading posts, which allow for the flexible membrane of the Tissue Train plates to deform around them and expose the 3D culture to equibiaxial strain.
2. Apply a thin layer of silicone grease to the top of the loading posts.
3. Place red gaskets onto the bottom of each 6-well plate.
4. Insert the prepared plates into the baseplate on top of the loading posts.
5. Move this apparatus into a 37 °C, humidified incubator with 5% CO₂ and connect the quick-disconnect fittings to the baseplate. Repeat steps 5.1 to 5.6, but omitting the connections to create a non-strained control.
   NOTE: Optionally, one can apply a tube sealing stopper, which prevents vacuum pressure application to the bottom of a single BioFlex well, allowing the generation of a non-strained control culture on the same plate.
6. Cover with a Plexiglas sheet and add ~5-10 lbs of weight to ensure a tight seal.
7. Turn on the air compressor and open the outflow valve from the air compressor.
8. Turn on the vacuum pressure pump and open the pressure reservoir valve connected to the vacuum tank outflow. Ensure that the vacuum pressure is around 90 kPa.
9. Open the inflow valve from the air compressor located near the bioreactor controller. Adjust the pressure so it is no more than 15 PSI at the vent port.
10. Launch the onboard software and set up the desired cyclic mechanical strain parameters. For this study, a regimen of 18% strain amplitude at 0.4 Hz for 24 h was used. To configure this, create a new regimen, set the minimum elongation to 0%, the maximum elongation to 18%, and start the protocol.
11. Observe the plate and the membrane to ensure that movement is visible throughout the strain session. The membrane should be deforming around the loading posts.
12. Check the water trap for moisture periodically, and observe the computer screen for run time, progress, and appropriate morphology of the strain regimen.
13. Disconnect the baseplate assembly upon completion of the strain regimen, and return the strained cultures to incubate for 24 h prior to downstream characterization analyses.
14. Power down and disassemble the system in reverse order of startup: close or turn off the air compressor inflow valve, then the pressure reservoir valve, the vacuum pressure pump, the air compressor outflow valve, and finally the air compressor, in that order.
15. Collect the supernatant after 24 h of incubation following exposure to mechanical strain. Store at -80 °C until use.

6. Morphological characterization of organoids via immunofluorescence

1. Wash the organoid-collagen gel model three times with PBS by gently adding 1 mL PBS, incubating for 5 min at room temperature.
2. Carefully aspirate the PBS and repeat the washing process a total of three times.
3. Fix the model by incubating 2 mL of 4% paraformaldehyde for 1 h at 4 °C.
4. Wash the models three times with PBS as described above.
5. Use a scalpel to gently cut the circular foam anchors in the flexible wells with the collagen gel attached and place it into a Petri dish.
6. Permeabilize the organoids by incubating with 2 mL of 0.1% Triton X-100 in PBS for 5 min at room temperature.
7. Wash the models three times with PBS.
8. Block non-specific binding by incubating the models with 3 mL of 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Remove the blocking solution and proceed to the next step.
9. Incubate the models overnight at 4 °C with a staining cocktail containing anti-human zonula occludens-1 (ZO-1) conjugated with Alexa Fluor 594 at 1:100 dilution, phalloidin conjugated with Alexa Fluor 488 at 1:1000 dilution for assessment of F-actin, and the nuclear stain, DAPI at 1:4000 dilution. The diluent is 1 mL of 1% BSA in PBS. Tape the edges of the plate prior to incubation at 4 °C.
10. Aspirate the staining cocktail solution the following day and wash the models three times with PBS.
11. Image using a confocal microscope and take Z-stacks at 5 µm slices of representative organoids at 40x objective magnification.
12. Analyze fluorescence intensity using Fiji image analysis software. Normalize the mean fluorescence intensity with the area of the organoid to ensure accurate comparison across different environmental conditions.

7. Functional characterization of organoids through ELISA and lactate dehydrogenase (LDH) cytotoxicity assay

1. Thaw supernatants on ice and assess for interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion using commercially available ELISA kits according to the manufacturer's protocol.
2. Assess for LDH release, a marker of cytotoxicity, using a commercially available LDH absorbance assay.
3. Generate a standard curve using recombinant LDH protein at half-log concentrations from 0.5 µg to 0.5 ng. Interpolate LDH concentrations from each sample using the standard curve.

8. Optional: Isolation of samples for RNA or protein analyses

1. Instead of adding 4% paraformaldehyde for immunofluorescence staining, add prepared RLT buffer containing β-mercaptoethanol at 10 µL per 1 mL of RLT.
2. Use 300 µL per model and pipette up and down to detach and lyse cells completely.
3. Transfer the lysate to an RNase-free microcentrifuge tube and place the tubes on dry ice to freeze quickly. Transfer to -80 °C for long-term storage.
   NOTE: Alternatively, one can use RIPA buffer for protein extraction by preparing: 50 mM Tris-HCl, pH 7.4-7.5; 150 mM NaCl; 1% NP-40 or Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS in deionized water. Premade RIPA buffer is also commercially available.
4. Add 200 µL per model, rock on ice for 5 min, then collect the lysate.
5. Spin at 15,000 x g for 10 min at 4 °C to pellet cell debris and transfer to -80 °C for long-term storage.

Results

Cell line-derived alveolar epithelial-fibroblast organoids were established and cultured for over 21 days. Morphological changes were observed throughout this time, with organoid number, area, and diameter assessed on days 3, 9, and 21 to monitor growth and development. The average number of organoids per well significantly decreased by days 9 and 21 compared to day 3 (Figure 2A). In contrast, the average organoid area and diameter increased significantly on days 9 and 21, indicating continu...

Discussion

Three-dimensional (3D) alveolar epithelial-fibroblast organoids represent a highly physiologically relevant platform for modeling lung structure and function under mechanical strain. Unlike 2D or even 3D co-culture systems, organoids preserve critical features of the in vivo lung microenvironment, including spatial organization, epithelial-mesenchymal crosstalk, and extracellular matrix (ECM) interactions¹⁴. Mechanical strain plays a central role in airway remodeling in diseases such as a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	Eppendorf	0030 125.215
15 mL conical tube	Fisher Scientific	14-959-53A
16% paraformaldehyde	Thermo Fisher Scientific	28908
2-mercaptoethanol	Millipore Sigma	M3148
50 mL conical tube	Fisher Scientific	14-432-22
6-well (flexible membrane) Tissue Train circular foam plate	Flexcell International	TTCF-5001
A549 cells	American Type Culture Collection (ATCC)	CCL-171
Air compressor	California Air Tools	10020DSPCAD
Bovine serum albumin	Millipore Sigma	A9418
Buffer RLT	Qiagen	79216
CO2 Incubator	PHCBI	MCO-170AICUVL-PA
Cultrex organoid harvesting solution	Bio-Techne	3700-100-01
CyQUANT LDH cytotoxicity assay kit	Thermo Fisher Scientific	C20300
DAPI (4′,6-diamidino-2-phenylindole)	Thermo Fisher Scientific	D1306
Dimethyl sulfoxide	Sigma Aldrich	D2650-100ML
Dulbecco’s modified Eagle’s medium (DMEM)	Thermo Fisher Scientific	11965092
EVOS XL Core microscope	Thermo Fisher Scientific	AMEX1200
Fetal bovine serum (FBS)	Thermo Fisher Scientific	A5209501
Fiji image analysis software	National Institutes of Health
Flexcell FX-6000 Tension and Tissue Train system (includes Flexlink FX-6000 Tension controller, pressure reservoir, baseplate, acrylic window, 25 mm diameter cylindrical Loading Stations, water trap, FlexSoft FX-6000 V1.0 software, and various tubing)	Flexcell International	FX6000T
Human Interleukin-6 Duoset ELISA	Bio-Techne	DY206
Human Interleukin-8 Duoset ELISA	Bio-Techne	DY208
Leica DMi8 confocal microscope	Leica Microsystems	11889122
Matrigel Basement Membrane Matrix	Corning	356237
MRC-5 cells	ATCC	CCL-185
Parafilm	Millipore Sigma	P7793
Penicillin/streptomycin (Pen/Strep)	Thermo Fisher Scientific	15140122
Phalloidin labeling probes, Alexa Fluor 488	Thermo Fisher Scientific	A12379
Phosphate buffered saline (PBS)	Thermo Fisher Scientific	14190-144
Rat tail collagen I	Sigma-Aldrich	C3867
Recombinant human lactate dehydrogenase protein (active)	Abcam	Ab93699
RIPA lysis buffer	Thermo Fisher Scientific	89901
RNase-free microfuge tubes	Thermo Fisher Scientific	AM12400
T75 cm2 culture flask	Greiner	658175
Triton X-100	Sigma-Aldrich	X100-5ML
ZO-1 monoclonal antibody (ZO1-1A12), Alexa Fluor 594	Thermo Fisher Scientific	339194