Generating an Ex Vivo Ovine Wounded Skin Model Infected with a Bacterial Pathogen

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

1. Sterilization

1. Disinfect forceps prior to collection of the heads by taking clean forceps and performing dry heat sterilization in an oven at 200 °C for 1 h. Autoclave all glassware at 121 °C for 15 min before use.
2. Carry out all described work within a microbiology class 2 cabinet. Prepare all reagents as per the manufacturer's instructions.

2. Sample collection

1. In the abattoir, Swaledale lambs were slaughtered by stunning using electricity or a captive-bolt pistol and exsanguinated. Collect lamb heads no more than 4 h post-slaughter.

3. Preparation of the heads

1. Disinfect the forehead section of the lamb by pouring approximately 100 mL of 200 ppm chlorine dioxide solution onto the sample area. Shave the forehead section of the head using electric clippers and wash the area with 200 mL of 200 ppm chlorine dioxide solution.
2. Wipe the area with ethanol and blue roll and cover the sample area with hair removal cream for 35 min. Gently scrape off the hair removal cream using a scraping tool and assess the sample area. If a significant amount of hair remains, repeat the hair removal process.
3. Use a further 200 mL of chlorine dioxide solution to rinse the area, and then rinse with ethanol and wipe with a blue roll.
4. Using a sterile 8 mm biopsy punch, cut out 8 mm skin samples from the prepared area. Remove the samples using sterile forceps and a 15-blade scalpel, ensuring that all cutaneous fat is removed.
5. Place the samples in a sterile 0.5 L jar filled with sterile phosphate-buffered saline (PBS), and then transfer them to sterile 50 mL tubes with 50 mL of 200 ppm chlorine dioxide solution, invert twice, and leave to sterilize for 30 min.
6. Remove the samples from the chlorine dioxide solution and wash them by placing them in a 50 mL tube filled with 40 mL of sterile PBS. Once washed, place each individual skin sample in a separate well of a 24-well plate.
7. Add 350 µL of pre-warmed medium while maintaining the sample at the air-liquid interface. The composition of the media is as follows: MK media (Medium 199 with Hanks' salts, L-glutamate, and 1.75 mg/mL sodium bicarbonate) and Ham's F12 in a 1:1 ratio, with added fetal bovine serum (FBS, 10% v/v), epidermal growth factor (EGF, 10 ng/mL), insulin (5 µg/mL), penicillin-streptomycin (100 U/mL), and amphotericin B (2.5 µg/mL).
8. Seal the 24-well plates with a gas permeable plate seal and incubate at 37 °C in a humidified 5% CO₂ tissue incubator for up to 24 h.

4. Maintenance of skin samples

1. After incubation, remove the culture medium and rinse the samples in 500 µL of sterile phosphate-buffered saline (PBS). Add antibiotic-free media to each sample and incubate at 37 °C in a humidified 5% CO₂ tissue incubator for 24 h to remove residual antibiotics in the sample.
2. If turbidity or fungal infection develop in the antibiotic-free media after 24 h, then discard the sample.

5. Preparation of the inoculum

1. Prepare a 50 mL tube with 10 mL of sterile tryptic soy broth. Take a fresh agar plate of S. aureus and use a swab to transfer several colonies into the broth. Incubate for 18 h at 37 °C at 150 rpm.
2. Centrifuge at 4,000 x g for 3 min. Remove the supernatant and resuspend the cell pellet in 10 mL of sterile PBS. Repeat twice to ensure adequate washing of the cells.
3. Adjust the inoculum to 0.6 OD₆₀₀ in sterile PBS. Confirm the inoculum load by undertaking a manual viable plate count.

6. Infection of skin samples

1. Prepare a fresh 24-well plate with 400 µL of pre-warmed antibiotic-free media and add in the 24-well inserts using sterile forceps.
2. Remove the media from the skin samples, wash with 500 µL of sterile PBS, and remove the wash. Use sterile forceps to gently hold the sample to the bottom of the well.
3. Use a 4 mm punch biopsy to make a central wound flap, piercing through to a rough depth of 1-2 mm. Then, use a 15-blade scalpel and sterile toothed allis tissue forceps to remove the top layer of the wound flap. Variability in the wound dimensions may affect the outcome of the infection and the endpoint colony forming units (CFU).
4. Once all the samples have been wounded, transfer them to the 24-well inserts using sterile forceps. Pipette 15 µL of the bacterial inoculum into the wound bed. Then, incubate for 24 h at 37 °C in a humidified 5% CO₂ tissue incubator.
5. If longer incubation periods are necessary, remove the media and replace it with fresh media every 24 h and incubate in the same conditions.