What type of mouse is used for this protocol?

B6 transgenic mice are used for harvesting tongue tissue.

What enzymes are included in the digestion cocktail?

The cocktail includes collagenase, hyaluronidase, and deoxyribonuclease.

How is the enzymatic reaction stopped?

The reaction is stopped by adding 5% FBS in PBS.

What is the purpose of filtering the cell suspension?

Filtering removes undigested tissue fragments and debris.

What is the incubation temperature for the digestion process?

The incubation temperature is 37 degrees Celsius.

How long should the cells be incubated in culture?

Cells should be incubated until distinct colonies are formed.

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

JoVE Encyclopedia of Experiments

Cancer Research

0 views • 3:24 min • April 30th, 2023

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The tongue constitutes stratified squamous epithelium and an underlying layer of dense connective tissue termed the lamina propria. The epithelium consists of multi-layered squamous epithelial cells that provide protection against mechanical stress.

To isolate epithelial cells, begin with a freshly harvested mouse tongue. Mince the tongue to obtain smaller tissue fragments. Next, treat the fragments with a customized digestion cocktail containing collagenase, hyaluronidase, and deoxyribonuclease enzymes.

Collagenase and hyaluronidase enzymes digest the extracellular matrix that holds the tissue together, promoting the release of individual cells into the suspension. Deoxyribonuclease degrades any free DNA released from lysed cells.

Add serum-containing buffer to the digested sample to inactivate the protein-hydrolyzing enzymes. Now, filter the sample through cell strainers to remove any undigested tissue fragments and debris and obtain a single-cell suspension of epithelial cells.

Centrifuge to separate the cells. Discard the supernatant containing the digestion mix. Resuspend the cell pellet in fresh media. Finally, transfer the desired volume of this cell suspension into a sterile culture dish. Incubate the culture.

The epithelial cells attach to the bottom surface of the culture dish. Depending on the proliferative capability of individual epithelial cells, they start forming colonies.

Begin by using surgical scissors to harvest the tongue from a 6-week-old male or female B6 transgenic mouse. Use a scalpel to mince the tissue into very small fragments and collect the pieces into a 15-milliliter tube containing 4.5 milliliters of RPMI plain medium without serum. Add 200 microliters of triple enzyme mix to the tube and incubate the sample at 37 degrees Celsius for 30 minutes, tapping the tube every 10 minutes to enhance the enzymatic dissociation of the tissue. At the end of the incubation, stop the reaction with 5% FBS in PBS, and filter the cell suspension through a 70-micrometer mesh strainer to separate the cells from the larger tissue fragments.

Collect the cells by centrifugation and resuspend the pellet in 3 milliliters of complete medium. Then, plate the cells in 3 milliliters of complete medium per 60-millimeter dish. Transfer cell aggregates retained on top of the filter to a separate 60-millimeter culture dish and add 3 milliliters of complete medium. Keep both dishes in the incubator until distinct cell colonies are formed.