This study outlines a method for cultivating colonic epithelial cells from stem cell cultures to create a functional intestinal barrier model. The process involves using collagen-coated well inserts and specific culture conditions to promote cell proliferation and differentiation.
Begin with a multi-well plate containing a collagen-coated well insert.
Add an expansion medium to the well.
The nutrients in the medium promote cell proliferation, while the differentiation inhibitors maintain a stem-like state.
Add a suspension of epithelial cell clusters derived from colon stem cell cultures to the well insert and incubate.
The cells adhere to the collagen-coated surface and receive nutrients from both the upper and lower compartments.
The inhibitors prevent cell differentiation and facilitate the proliferation of the cell clusters.
Using a phase-contrast microscope, observe the culture.
Once the monolayer is formed, replace the culture medium with fresh medium devoid of cell differentiation inhibitors.
This facilitates the differentiation of the cells into mature colonic epithelial cells with tight junctions.
These cells secrete mucus and develop a functional monolayer that mimics the physiological intestinal barrier for host–pathogen interaction studies.
Pipette 600 microliters of expansion medium into the space beneath each insert. Pipette 100 microliters of cell suspension from the vial into each insert.
Return the plate to the tissue culture incubator and leave undisturbed for at least 12 hours. Avoid shaking or sharply tilting the plate. After incubating, place the plate on a phase contrast light microscope with a 2.5 times to 10 times objective lens.
Refresh with culture medium without inhibitors to discontinue inhibition treatment. Continue to refresh the medium every two to three days until confluent.
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