A Minimally Invasive Transthoracic Injection Technique for Reproducible Intrapleural Delivery in Mice
Abstract
1. Introduction
2. Experimental Design
2.1. Materials
- RPMI 1640, HEPES (ThermoFisher Scientific, Merelbeke, Belgium, Cat. Number 52400025)
- Fetal Bovine Serum (ThermoFisher Scientific, Cat. Number 10270–106)
- L-Glutamine (ThermoFisher Scientific, Cat. Number 25030024)
- TrypLE Express Enzyme (ThermoFisher Scientific, Cat. Number 12604–021)
- Dulbecco’s Phosphate Buffered Saline (ThermoFisher Scientific, Cat. Number 14190–144)
- Trypan Blue solution 0.4% (ThermoFisher Scientific, Cat. Number 15250–061)
- BD Plastipak™ 1 mL luer slip syringes, concentric (VWR, Leuven, Belgium, Cat. Number 613–5399)
- BD Microlance™ 3 needles, 27 G, 13 mm long (VWR, Cat. Number 613–3832)
- 200 µL pipette tips (Sarstedt, Antwerp, Belgium, Cat. Number 70.3030)
- Azpack™ Carbon Steel Razor Blades (Fisher Scientific, Merelbeke, Belgium, part of ThermoFisher Scientific, Cat. Number 11904325)
- Soft-Zellin-C alcohol cleansing pads
2.2. Limitations
3. Procedure
3.1. Cell Suspension Preparation
- AE17 mesothelioma cells are cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) and 2 mM of L-glutamine. After thawing, cells must be passaged at least twice but no more than five times before inoculation.
- Detach cells from the culture flask TrypLE and wash them twice in sterile phosphate-buffered saline (PBS). Count the cells and resuspend them in an appropriate volume of sterile PBS to achieve the desired final concentration.
- Assess cell viability using the trypan blue exclusion assay, ensuring a viability of >95% before injection.
3.2. Needle Preparation
- Assemble a 0.3 mL or 1 mL syringe with a 27 G needle. Place a 100–200 µL (yellow) pipette tip over the needle, marking the pipette tip approximately 2.5 mm below the needle point.
- Using a straight razor blade, cut the pipette tip at the marked location.
- NOTE: Needle lengths may vary slightly. It is advisable to prepare multiple pipette tips of varying sizes to ensure a proper fit. Select the correct pipette tip length for each needle assembly.
- Fill the syringe with sufficient cell suspension for 100 µL per mouse. It is recommended that a fresh needle is used for each animal.
- Reassemble the cut pipette tip onto the needle, ensuring that no more than 2.5 mm of the needle tip is exposed. Secure the pipette tip in place with autoclave tape if necessary.
3.3. Transthoracic Injection
- Restrain the mouse by firmly scruffing it. Ensure that its front limb (right for right-handed handlers, left for left-handed handlers) is lifted and immobilised. The skin should be pulled taut over the ribcage.
- Disinfect the injection site with an ethanol wipe and part the fur to expose the skin. Identify the intercostal space below the axilla (avoiding the fat pad in the armpit).
- Insert the needle at the selected injection site with the bevel facing downward. Ensure the pipette tip is flush against the skin, applying gentle pressure.
- Slowly inject 100 µL of the cell suspension. After injection, release the mouse and observe it for a few minutes to monitor for signs of pneumothorax (e.g., laboured breathing, signs of distress). If pneumothorax occurs, euthanise the mouse immediately.
3.4. In Vivo Monitoring
- Monitor body weight and general health throughout the experiment. See Expected Results for tumour kinetics.
- Potential clinical symptoms include respiratory distress (e.g., shortness of breath), weight gain due to fluid accumulation (ascites), lethargy, and sudden weight loss. Mice should be euthanised if at least two of these symptoms are observed.
3.5. Pleural Fluid Collection
- To collect pleural fluid, the animal must lay flat on its back after euthanasia and four paws should be pinned to the work surface area.
- Make a small transverse incision, approximately 1 cm, on the abdomen using a sharp pair of scissors followed by a vertical anterior incision along the midline up to the top of the mediastinum. Open the skin from both sides. Then perform similar transverse incision in the peritoneal wall. The anterior incision of the peritoneal wall should only be large enough to expose the liver/stomach. It is very important to keep the pleural cavity intact at this stage.
- Using a pair of tweezers, carefully move back the liver to expose the entire diaphragm and visualise the effusion.
- Carefully insert a 25 G needle into the pleural space through the diaphragm, where the effusion has accumulated. Gently collect the fluid in a syringe (the needle might need to be repositioned at times). The fluid can then be transferred into an appropriate tube and stored accordingly for further analysis.
- OPTIONAL STEP: If little or no spontaneous effusion is present, the user may wish to perform a lavage, which can be performed with up to 1 mL of balanced salt solution or standard tissue culture media. Perform steps 1 to 4 as described above by injecting the desired solution into the closed chest cavity through the diaphragm prior to withdrawing the fluid through the same injection site.
3.6. Tumour Collection
- After collecting pleural fluid, circumferentially dissect the diaphragm to detach it. Carefully inspect the diaphragm and collect any tumours growing on its surface.
- Perform a median sternotomy by cutting along both sides of the sternum and removing it. This exposes the heart, lungs, and thymus. Inspect the thoracic cavity thoroughly for tumours, which may be present on the chest wall, lungs, heart, thymus, or diaphragm. Tumours typically appear light in colour, smooth or slightly nodular, and firm.
- Excise all visible tumours and process them for embedding and further histological analysis as required.
4. Expected Results
4.1. Validation of Injection Accuracy
4.2. Post-Injection Weight Monitoring Reveals Variable Physiological Responses
4.3. Macroscopic Examination Confirms Tumour Development in Injected Mice
4.4. Histological Analysis Demonstrates Progressive Tumour Growth
5. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
FBS | Foetal bovine serum |
H&E | Haematoxylin and eosin |
PBS | Phosphate-buffered saline |
PDOX | Patient-derived orthotopic xenograft |
PM | Pleural mesothelioma |
SOI | Surgical orthotopic implantation |
Appendix A
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Rovers, S.; Farahmand, P.; Liu, D.; Brants, L.; Hermans, C.; Peeters, D.; McKinven, D.; Doig, J.; Lardon, F.; van Meerbeeck, J.; et al. A Minimally Invasive Transthoracic Injection Technique for Reproducible Intrapleural Delivery in Mice. Methods Protoc. 2025, 8, 55. https://doi.org/10.3390/mps8030055
Rovers S, Farahmand P, Liu D, Brants L, Hermans C, Peeters D, McKinven D, Doig J, Lardon F, van Meerbeeck J, et al. A Minimally Invasive Transthoracic Injection Technique for Reproducible Intrapleural Delivery in Mice. Methods and Protocols. 2025; 8(3):55. https://doi.org/10.3390/mps8030055
Chicago/Turabian StyleRovers, Sophie, Pooyeh Farahmand, Dana Liu, Louize Brants, Christophe Hermans, Dieter Peeters, Danielle McKinven, Jennifer Doig, Filip Lardon, Jan van Meerbeeck, and et al. 2025. "A Minimally Invasive Transthoracic Injection Technique for Reproducible Intrapleural Delivery in Mice" Methods and Protocols 8, no. 3: 55. https://doi.org/10.3390/mps8030055
APA StyleRovers, S., Farahmand, P., Liu, D., Brants, L., Hermans, C., Peeters, D., McKinven, D., Doig, J., Lardon, F., van Meerbeeck, J., Marcq, E., Murphy, D. J., & Smits, E. (2025). A Minimally Invasive Transthoracic Injection Technique for Reproducible Intrapleural Delivery in Mice. Methods and Protocols, 8(3), 55. https://doi.org/10.3390/mps8030055