Pig Bronchiolar Tissue Extraction: A Procedure to Dissect Bronchiolar Tissue Sections From Porcine Lungs

Samenvatting

Source: Harrington, N. E. et al. Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung. J. Vis. Exp. (2021)

In this video, we describe a surgical procedure to extract bronchiolar tissues from harvested pig lung. The ex vivo bronchiolar tissue could be used to develop infection models for studying chronic lung infections.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dissection of ex vivo pig lung (EVPL) tissue

1. Obtain lungs from the designated source as soon as possible after slaughter, ensuring they are kept cold by transporting to the laboratory in a domestic cool box.
   NOTE: Lungs closer to the day of slaughter show less bruising from storage, but tissue kept on cold storage for up to 4 days from slaughter can also be used. As the cool box needs to be taken into the butcher's shop or abattoir, it must be decontaminated following local lab guidelines after each use and stored outside the microbiology lab when not in use, to reduce the risk of contamination and a breach of containment.
2. Working on a sterilized surface and under a flame, place the lungs on a clean plastic chopping board covered with autoclaved aluminum foil. Check that the bronchioles remain intact. If there has been any damage at the abattoir or during transport the lungs are not suitable for use.
3. Heat a palette knife under a flame and very briefly touch the knife to the area of the lung surrounding the bronchiole to sterilize the surface of the tissue.
4. Cut away the surface tissue surrounding the bronchiole using a sterile mounted razor blade. Make incisions parallel to the bronchiole to prevent any damage.
5. Once the bronchiole has been exposed, make a cross-sectional incision through the bronchiole at the highest point visible to free the bronchiole.
6. Using sterile forceps, lightly hold the free end of the bronchiole and cut away any remaining unwanted tissue using a sterile mounted razor blade. Make a final cross-sectional incision across the bronchiole before any branching is visible to remove the bronchiole from the lungs.
7. Place the bronchiole in the first DMEM/RPMI 1640 wash. Leave the bronchiole in the wash and repeat steps 1.2-1.5 to harvest additional sections of bronchiole from the same lung as required to yield sufficient tissue sections.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
70% ethanol or similar for surface sterilizaton and flamin gof dissection equipment
Aluminum foil - pre-sterilised by autoclaving - to cover the chopping board on whcih you wil dissect lungs
Bunsen burner
Chopping board - we recommend a plastic board to allow for easy decontamination with alcohol.
Coolbox to transport lungs to lab
Dissection scissors in different sizes
Dulbecco's modified Eagle medium (DMEM)
Fisherbrand 2 mL reinforced tubes	Thermo Fisher	15545809
Large pallet knife
Mounted razor blades
Plastic chopping board and aluminium foil to create a sterile and cleanable dissection surface
Roswell Park Memorial Institute (RPMI) 1640 medium
Selection of forceps (blunt tips recommended)
Suitable containers for disposing of contaminated sharps and pig ung tissue, according to your institution's health & safety policies.