Fabrication of Extracellular Matrix from Mouse Model: An Effective Procedure to Prepare Decellularized Tongue Extracellular Matrix from Murine Tongue

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

1. Preparation of Tongue Extracellular Matrix (TEM)

1. Execute mice by cervical dislocation and remove the tongues using sterile surgical scissors and tweezers.
2. Immerse the tongues in 75% ethanol for 3 min, then put each tongue into a 1.5 mL Eppendorf (EP) tube with 1 mL of 10 mM sterile phosphate-buffered solution (PBS).
   NOTE: The concentration of PBS in all the following steps is the same as the concentration in this step.
3. Cell lysis by freeze-thaw: Freeze the tongues in EP tubes at -80 °C for 1 h and then thaw the tongues at room temperature for 45 min for 3 cycles.
4. Load each tongue onto a piece of surgical suture using a surgical needle and wrap the end of the suture with a small piece of sterile tinfoil. Perform the operation in a 3.5 cm or 6 cm culture dish containing 75% ethanol in sterile conditions.
   NOTE: The appropriate length of each piece of surgical suture is about 20 cm, and the appropriate size of each piece of tinfoil is about 0.3 cm² (1 cm x 0.3 cm). The tongue should be loaded near the tinfoil.
5. Rinse each tongue with 3 mL of sterile PBS in a 3.5 cm or 6 cm culture dish for 30 s. Perform this operation in sterile conditions.
6. Wash the tongues with ultrapure water: Add ampicillin into a wide-mouth bottle with 250 mL of sterile ultrapure water to a final concentration of 90 µg/mL. Put the tongues into the bottle containing a stir bar. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.
   NOTE: Up to 5 tongues can be put into the same bottle in consideration of the twining of the suture. The tinfoil is at the end of the suture in the bottle to prevent the tongue from slipping off. The tongues should be placed 2 cm high from the bottom of the bottle by adjusting the length of the suture remaining inside the bottle. This note is also for steps 1.8, 1.10, 1.12, and 1.16.
7. Put the bottle on a magnetic stirrer for 12 h.
8. Wash the tongues with NaCl: Add ampicillin into a wide-mouth bottle with 250 mL of sterile 1 M NaCl to a final concentration of 90 µg/mL. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.
9. Put the bottle on a magnetic stirrer for 24 h.
10. Cell lysis by Triton X-100: Add ampicillin to a final concentration of 90 µg/mL into a wide-mouth bottle with 250 mL of sterile 2% Triton X-100 in PBS. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.
11. Put the bottle on a magnetic stirrer for 48 h.
12. Wash tongues with CaCl₂/MgCl₂: Add ampicillin into a wide-mouth bottle with 250 mL of sterile 5 mM CaCl₂/MgCl₂ to a final concentration of 90 µg/mL. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.
13. Put the bottle on a magnetic stirrer for 24 h.
14. Digestion by DNase: Add 1 mL of Hank's balanced salt solution (HBSS) to each EP tube. Add DNase into HBSS respectively to a final concentration of 300 µM. Move each tongue into each EP tube, with part of the suture outside the tube. Perform this operation in sterile conditions.
    NOTE: Make sure that the part of the suture that remains inside the bottles in the previous steps also remains inside the EP tube in this step, and make sure that the part of the suture that remains outside the bottles in the previous steps also remains outside the EP tube in this step.
15. Incubate the tongues in EP tubes at 37 °C for 24 h.
16. Wash the tongues with PBS: Add ampicillin into a wide-mouth bottle with 250 mL of sterile PBS to a final concentration of 90 µg/mL. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.
17. Put the bottle on a magnetic stirrer for 24 h.
18. Store the prepared TEM in sterile PBS at 4 °C until use.