Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Samenvatting

Source: Shechtman, L. A. et al. Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells. J. Vis. Exp. (2021)

In this video, we describe the isolation of circumvallate papillae (CVP) epithelium from a murine tongue.

Protocol

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

1. Isolation of CVP Epithelium

1. Euthanize the mice with CO₂ asphyxiation according to IACUC regulations, followed by an approved secondary method such as bilateral thoracotomy, cervical dislocation, decapitation, or exsanguination.
2. Use large sterile dissection scissors to cut the cheeks and break the jaw. Lift the tongue and cut the lingual frenulum to separate the tongue from the floor of the oral cavity. Cut out the tongue and collect it in sterile ice-cold Dulbecco's phosphate-buffered saline (dPBS) with Ca²⁺ and Mg²⁺.
3. Remove and discard the anterior tongue by cutting just anterior of the intermolar eminence with a razor blade (Figure 1A, dashed line). Use a delicate task wipe to remove any hair and excess liquid from the posterior tongue.
4. Fill 1 mL syringe with 200–300 µL of injection enzyme solution (final concentration: 2 mg/mL type-I Collagenase and 5 mg/mL Dispase II in Ca²⁺/Mg²⁺-containing dPBS, diluted from 10 mg/mL stock solutions) and insert a 30 G x 1/2 needle just above the intermolar eminence (Figure 1B, black arrow) until just anterior to the CVP (Figure 1B, black box). Inject the enzyme solution underneath and at the lateral edges of the CVP between the epithelium and the underlying tissues (lamina propria, muscle). Withdraw the syringe slowly and continuously from the tongue as the injection is performed.
5. Incubate the tongue in sterile Ca²⁺/Mg²⁺-free dPBS at room temperature for precisely 33 min.
6. Make small cuts in the epithelium bilaterally and just anterior to the CVP using extra fine dissection scissors, and gently peel the epithelium by lifting it with fine forceps. Once the trench epithelium is free of the underlying connective tissue, place it in an empty 2 mL microcentrifuge tube pre-coated with FBS. Do the epithelial trimming before or after detaching the CVP epithelium (Figure 1C, D).

Resultaten

Figure 1: Dissected tongue and peeled CVP epithelium. (A) The tongue is dissected out, and the anterior tongue is removed by cutting ju...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
1 mL TB Syringe	BD Syringe	309659
Dulbecco’s Phosphate Buffered Saline with Ca²? & Mg²?	Gibco	10010-023
Dulbecco’s Phosphate Buffered Saline with Ca²? & Mg²?	Sigma Life Sciences	D8662-500ML
Dumont #5 Forceps	Fine Science Tools	11252-30
Fetal Bovine Serum (FBS)	Gibco	26140-079
1 mL TB Syringe 	BD Syringe	309659
2.0 mL Microcentrifuge Tubes 	USA Scientific 	1420-2700
Surgical Scissors - Sharp	Fine Science Tools	14002-14
Extra Fine Bonn Scissors 	Fine Science Tools 	14084-08
Big-Digit Timer/Stopwatch	Fisher Scientific	S407992
Stereomicroscope	Zeiss	Stemi SV6
Cohan-Vannas Spring Scissors	Fine Science Tools	15000-02
Collagenase from Clostridium histolyticum, type I	Sigma Life Science	C0130-1G
Dispase II (neutral protease, grade II)	Sigma-Aldrich (Roche)	4942078001