Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching
June 30th, 2010
Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.
In this video, a method for the dissection and culture of mouse embryonic lungs is demonstrated. The procedure begins with the harvesting of embryos at E 11.5 or E 12.5. Next, the embryos are opened and the lungs are extracted.
The lungs are then plated in a liquid air interface and incubated following culture. Changes in the growth and branching of mesen chy and epithelium of the embryonic lung in response to specific treatments can be observed using a stereo microscope. Hello, I'm David War Burton.
I direct the developmental biology and regenerative medicine program at the Ban Research Institute Children's Hospital, Los Angeles. And I'm Jan Carro, a senior postdoc in Dr.War Burton Lab. Today we are going to show you a technique for isolating embryonic mouse lung and placing them in organ culture.
This technique has evolved from the original description by Tina Jaco Halkin and Pablo bring us at the USC School of Dentistry about 20 years ago. We use this procedure in our lab to study lamb branching morphy. So let's get started.
On Postum Day 11.5 or 12.5, place a carbon dioxide ssis sacrificed timed pregnant mouse under a laminar flow hood on its back. Spray with 70%ethanol. Make an incision in the abdomen.
Then while holding the animal's hind legs, remove the skin by pulling upwards. Open the peritoneal cavity and remove the uterus. Place the uterus in a 50 milliliter conical tube filled with cold hanks balanced salt solution or HBSS and gently rock for two minutes to rinse off the blood.
Next, to transfer the uterus to a Petri dish under a stereoscopic dissecting microscope. Using spring scissors, cut the uterine wall to release the embryos. Then using a perforated spoon, harvest the embryos and place them in a Petri dish containing HBSS and place on ice.
Continue working under the stereoscopic dissecting microscope and begin dissecting embryonic lungs. Begin by placing the embryo on its left side. Using forceps, remove the right forelimb holding forceps in the left hand.
Cut the embryo starting from the limb opening and remove the skin. This step will allow you to see internal organs and to localize the heart and lungs for the following step. Remove the heart.
The embryonic lungs are located posterior to the heart and anterior to the spine. Next, separate the intact lung from the embryo. Then trim away extraneous tissue and the esophagus.
Once the lungs have been removed, use a sterile transfer pipette to transfer them to a Petri dish containing cold HBSS on ice. To each well of a no clone delta polystyrene dish at 0.5 to one milliliter of D-M-E-M-F 12 supplemented with 50 units per milliliter of penicillin streptomycin. Next place a nucleo polycarbonate track etch membrane on top of the media, shiny side down and rough side up.
Using a sterile transfer pipette place a dissected embryonic lung on top of the nucleo polycarbonate track etch membrane. Using the stereo microscope, adjust the embryonic lung position. The embryonic lung should have an intact trachea placed in a straight position as shown here.
This ensures that all of the lungs have the same internal pressure, transfer the dish to a cell incubator and culture for the desired time. Here, E 12.5 lungs were grown in the presence or absence of FGF nine. FGF nine is a member of the fibroblast growth factor family that induces expansion of the mechy and the dilation of the epithelium in lungs grown in vitro after 48 hours.
In the absence of FGF nine, increased branching is seen. These images are photographs of the embryonic lung taken under a stereo microscope. In contrast, lungs grown 48 hours in the presence of FGF nine have dilated epithelium and mechy.
After 24 hours of culture. After 48 hours, the effect on the epithelium is even more pronounced. So we have just shown you how to prepare organ cultures from embryonic mouse lung.
When doing this procedure is important to remember to remove the the lung from the embryo as soon as possible to reduce the tissue deterioration. So thanks for watching. Hopefully you'll find this a useful technique.
Good luck with your experiments.
Overview
This article presents a method for isolating and culturing embryonic mouse lungs to study epithelial-mesenchymal interactions. The technique allows for the observation of growth and branching changes in response to specific treatments.
Key Study Components
Area of Science
- Developmental Biology
- Regenerative Medicine
- Organ Culture Techniques
Background
- Embryonic lung organ culture is crucial for studying lung development.
- Epithelial-mesenchymal interactions are key to understanding lung morphogenesis.
- The method allows manipulation of culture conditions.
- Previous techniques have evolved over the years for improved outcomes.
Purpose of Study
- To demonstrate a reliable method for isolating embryonic mouse lungs.
- To observe the effects of specific treatments on lung development.
- To provide a resource for researchers studying lung biology.
Methods Used
- Dissection of mouse embryos at E 11.5 or E 12.5.
- Isolation of embryonic lungs under a stereo microscope.
- Culture of lungs in a liquid air interface.
- Observation of growth and branching using microscopy.
Main Results
- FGF9 treatment leads to dilation of epithelium and mesenchyme.
- Increased branching is observed in the absence of FGF9.
- Changes in lung morphology can be documented over time.
- Proper timing in lung removal is critical to reduce tissue deterioration.
Conclusions
- The method is effective for studying embryonic lung development.
- Organ culture techniques can reveal important biological interactions.
- This approach can be utilized in various experimental settings.
Frequently Asked Questions
Executive Industry Relevance
This embryonic lung culture system enables mechanistic interrogation of epithelial-mesenchymal signaling pathways critical for lung branching morphogenesis. By providing a defined, serum-free platform to manipulate growth factors like FGF9, it supports target validation and de-risking in pulmonary therapeutic discovery. The model offers translational relevance for identifying modulators of lung development and repair.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables functional testing of mesenchymal-derived inductive signals on epithelial branching and morphogenesis.
- Operational Value: Supports gain- and loss-of-function studies using recombinant proteins, viral vectors, or RNAi to validate target specificity.
- Predictive Value: Quantifies structural outcomes such as epithelial dilation and mesenchymal expansion in response to pathway modulation.
Screening & Assay Development
- Assay Readiness: Establishes a reproducible liquid air interface culture system for consistent lung explant growth and branching analysis.
- Quantitative Output: Enables morphometric measurement of epithelial dilation, mesenchymal expansion, and branching frequency under stereo microscopy.
- <Scalability: Compatible with multi-well formats for parallel compound or genetic perturbation screening.
Translational & Preclinical Research
- Disease Relevance: Models early lung morphogenesis to inform congenital disorder mechanisms and regenerative strategies.
- Translational Continuity: Bridges discovery-stage pathway modulation with preclinical evaluation of lung repair or fibrosis interventions.
- Mechanistic De-risking: Clarifies epithelial-mesenchymal crosstalk to reduce ambiguity in target selection for pulmonary therapeutics.
Pipeline & Workflow Integration
The method fits within early discovery workflows where pathway-specific modulation is tested in a physiologically relevant, ex vivo organ system prior to in vivo validation.
- Discovery Biology: Facilitates hypothesis testing of fibroblast growth factor and other signaling pathways in lung development.
- Screening: Provides a standardized platform for assessing compound effects on lung morphogenesis with quantitative imaging readouts.
- Analytics: Generates morphological and spatial data enabling comparison of treatment conditions across experimental groups.
- Translational Research: Supports continuity from mechanistic insight to preclinical models of lung injury or developmental repair.
- Enterprise Reuse: Defined, serum-free conditions allow adaptation across labs and projects studying pulmonary morphogenesis or repair.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in lung development pathways through direct observation of epithelial-mesenchymal interactions.
- Operational Value: Ensures reproducibility via standardized dissection, culture, and imaging protocols under controlled conditions.
- Strategic Value: Improves target confidence by linking molecular perturbations to quantifiable structural outcomes in a developing organ.
- Portfolio Impact: Enables early de-risking of pulmonary targets by validating functional relevance in a human-relevant developmental system.
Implementation Considerations
- Requires expertise in embryonic dissection and sterile tissue handling under stereo microscopy.
- Depends on access to cell culture incubators, sterile transfer tools, and defined media components.
- Necessitates standardization of embryo staging (E11.5–E12.5) and lung isolation timing to minimize variability.
- Adaptation to alternative model systems may require optimization of dissection and culture conditions.
- Practical limitations include tissue viability window and dependence on precise embryonic staging.
