Mouse Lung Organoids: A Working Protocol From Dissection to Passage

Lung organoids are three-dimensional cultures that self-assemble from adult stem and progenitor cells inside a Matrigel scaffold. They reproduce far more of the native airway and alveolar architecture than any flat monolayer. That fidelity is also their weakness. The same steps that make the model physiologically relevant make it fragile. A few minutes of warm ischemia, an over-digested suspension, or a trapped bubble will silently change the outcome three weeks later. The protocol below states each decision point together with the numbers that matter.

Schematic of a 3D lung organoid culture workflow, showing tissue collection, Matrigel embedding, medium exchange, passaging, cryopreservation, and the three application areas of lung development, disease modelling, and drug screening.

Why the Lung Rewards a Written Procedure

Two features of lung tissue shape every step. The organ is gas-filled and mechanically delicate, so dissociation must be both gentle and quick. The alveolar niche also depends on a defined growth factor mix, which makes medium composition as large a variable as surgical technique. A written procedure removes operator-dependent drift and makes batch comparisons meaningful. Teams that skip it usually see the same pattern: clean cystic organoids in one pair of hands, debris in another, and no way to identify which variable moved. Standardization is not paperwork here. It is the assay.

The Reagent Bench

Eight items should be ready before the first animal is opened. The list covers Matrigel, tissue digestion solution, rinse solution, DPBS, red blood cell lysis buffer, organoid digestion solution, freezing medium, and complete medium. Every surface that will contact cells gets a rinse with the rinse solution first. Untreated plastic adsorbs cells, and that loss stays invisible until counting day.

Step One: Dissection That Preserves Viability

Work aseptically and keep every tube on ice. Euthanize the animal and disinfect with 75% ethanol. Fix the mouse supine and open the skin from abdomen to neck, then strip it back to expose the subcutaneous layer. Open the abdominal muscle and displace the liver and intestine to reveal the diaphragm. Cut the diaphragm carefully, sever the ribs along the lateral thoracic margin, and remove the sternum to expose heart and lungs. Cut the renal artery, lift out the intact lung, and transfer it to pre-chilled DPBS.

In the dish, separate the individual lobes and strip away connective tissue and blood clots. Wash three to five times with DPBS until the supernatant is clear. Rough handling at this stage costs epithelial cells that never appear in the final count.

Step Two: Digestion and the Endpoint Call

Transfer the cleaned tissue to a 1.5 mL tube and mince with sterile scissors to pieces of roughly 0.5–2 mm³; 1 mm³ is acceptable. Move the fragments into 2–5 mL of pre-warmed digestion solution. Incubate at 37 °C in a shaking incubator or incubator for 15–40 minutes, inspecting the tube repeatedly.

The endpoint is visual, not timed. Digestion is complete when most fragments look flocculent and the suspension contains many cell clusters. Stop the reaction with at least three volumes of serum-containing medium. Pass the suspension through a 100 μm strainer to remove undigested tissue, then centrifuge the filtrate at 250–300 g for 3–5 minutes at 4 °C.

A red pellet means red cells survived. Resuspend in red blood cell lysis buffer for 2 minutes at room temperature, stop with DPBS, and centrifuge again. Resuspend the final pellet in DPBS, count, and adjust to 5×10⁵–1×10⁶ cells/mL.

Step Three: The Matrigel Droplet

Cool the pipette tips and keep the Matrigel on ice. Combine the cell pellet with Matrigel at a 1:4 volume ratio and mix by gentle pipetting for no more than 30 seconds. Bubbles are the main enemy at this step; a single bubble expands in the incubator and disrupts the dome.

Place 40–50 μL of the mixture in the centre of each well of a 24-well plate, avoiding the wall. Invert the plate and incubate at 37 °C for 15–20 minutes until the droplet has fully polymerized.

Step Four: Feeding and the Maturation Window

Add 500 μL of complete medium, warmed to room temperature, slowly down the well wall so the dome is not disturbed. Culture at 37 °C with 5% CO₂ and replace the medium every 2–3 days. Under good conditions, mature organoids with a clear central lumen appear within 3–14 days. Record diameter and morphology at each feed; those notes are the earliest warning of a drifting culture.

Step Five: Passage, Freezing, and Recovery

Passage when organoids exceed 300–400 μm in diameter, or after roughly 14 days. Aspirate the old medium and add organoid digestion solution, incubating at 2–8 °C for about 30 minutes to dissolve the Matrigel. Pipette gently during the incubation. Collect the suspension and centrifuge at 250 g for 5 minutes at 4 °C, then wash twice with DPBS to remove residual matrix. Resuspend in complete medium and re-embed at a 1:4 to 1:6 split ratio.

For cryopreservation, choose cultures with the best morphology, typically after two or three passages or in log phase. Use a dedicated freezing medium with controlled-rate cooling, then transfer to liquid nitrogen. Thaw in a 37 °C water bath for 1–2 minutes and resuspend directly in Matrigel for plating. Leave the plate undisturbed for the first three days to let the cells recover.

Where the Protocol Usually Breaks

Product Information

The kit supplies the recombinant protein components that support lung organoid growth and differentiation. Building a complete medium from individual factors takes time and introduces lot-to-lot variation; a defined set removes both problems. It is intended for construction, expansion, and basic research on normal mouse lung tissue.

Conclusion

A lung organoid is the sum of many small decisions: how long the tissue sat on ice, when digestion was stopped, whether the droplet touched the wall. None of these is difficult on its own. Written down with numbers attached, they become a process that any trained operator can repeat. That reproducibility, not any single reagent, is what turns organoids into a usable model.

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