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.

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% peni....

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