Isolation and Culture of Murine Tongue Epithelial Cells: A Procedure to Isolate and Culture Tongue Epithelial Cells from Mouse Model

Overview

In this video, we describe the isolation of epithelial cells from a murine tongue. These cells can be further cultured to give rise to cell colonies.

Protocol

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

1. Isolation and Culture of Primary Cells

1. Euthanize a 6-week-old male or female B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J by CO₂ asphyxiation or any other IACUC approved protocol.
   NOTE: These CRISPR/Cas9 knockin mice have Cre recombinase-dependent expression of cas9 endonuclease and EGFP directed by a CAG promoter. The upstream Lox-Stop-Lox (LSL) sequence present in the genome of these mice prevents the expression of Cas9 and EGFP in the absence of Cre recombinase. When used in combination with single guide RNAs (sgRNAs) and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo.
2. Dissect the tongue from the euthanized mice using surgical scissors.
3. Manually dissociate the tissue by mincing the tongue tissue into very small fragments using a scalpel. Collect the tissue fragments in a 15 mL tube containing 4.5 mL of RPMI plain medium (without serum).
4. Add the triple enzyme mix (200 µL) (see Table 1 for the recipe) to the tissue fragments.
5. Incubate the tissue-enzyme mix at 37 °C for 30 min and tap the tube every 10 min to enhance enzymatic dissociation of the tissue.
6. Add 5% fetal bovine serum (FBS) containing HBSS/PBS to the tissue-enzyme mix to stop the enzyme action.
7. Filter the above cell suspension through a sterile 70 µm nylon mesh to separate the dispersed cells and larger tissue fragments.
8. Wash the filtered cell suspension by centrifugation for 300 x g for 5 min in HBSS/PBS at RT.
9. Resuspend the pellet in culture medium (10% FBS in RPMI/DMEM) and grow in 60 mm culture dishes until distinct cell colonies are formed.
   1. Culture cell aggregates are retained on top of the filter in a 60 mm culture dish containing 3 mL of complete media (10% FBS in RPMI/DMEM) until cell colonies are formed.
10. Microscopically examine the primary cells for the presence of fibroblast contamination after 1 week of culture. Treat the primary cells developed from aggregates and cell suspensions with 0.25 trypsin 0.02% EDTA solution at 37 °C for 1 min to remove fibroblasts.
    NOTE: Usually the primary culture from cell aggregates produces more colonies compared to single-cell suspensions. The cells from these colonies provide better transduction efficiency with AAV transduction.

10X Triple Enzyme Stock Solution:
Collagenase	1 g final conc. [10 mg/mL]
Hyaluronidase	100 mg [1 mg/mL]
DNase	20,000 Units final conc. [200 mg/mL]
PBS 1X	Make up to 100 mL

Table 1: Recipe of buffer used in this study

Materials

Name	Company	Catalog Number	Comments
Mouse strains
C57BL/6	Envigo
B6;129-Gt(ROSA)26Sortm1(CAGcas9*,-EGFP)Fezh/J	Jackson labs	24857
NOD.CB17-Prkdc-scid/NCr Hsd (Nod.Scid)	Envigo
Enzymes
Benzonase	Sigma	E1014
Collagenase IV	Thermo Fisher Scientific	17104019
DNAse	Thermo Fisher Scientific	18047019
Hyaluronidase	MilliporeSigma	H3506
Trypsin	Biological Industries Israel BeitHaemek Ltd.	03-050-1B
Culture Media, Chemicals and Reagents
BSA	Amresco	0332-TAM-50G
DMEM	Biological Industries Israel BeitHaemek Ltd.	01-055-1A
FBS	Biological Industries Israel BeitHaemek Ltd.	04-127-1A
HBSS	Sigma	H6648
PBS	Biological Industries Israel BeitHaemek Ltd.	02-023-1A
Tris buffer	MERCK Millipore	648311-1KG
Plastic wares
Amicon-ULTRA filter 100 KDa	Millipore	UFC910024
0.22 µm sterile filters, 4 mm	Millex	SLGV004SL
0.45 µm sterile filters, 13 mm	Millex	SLHV013SL
Culture plates	Greiner Bio-One
Falcon tubes	Greiner Bio-One