What are the key components of the culture medium?

The culture medium includes nutrients for cell proliferation and differentiation inhibitors to maintain a stem-like state.

How long should the cells be incubated?

Cells should be incubated undisturbed for at least 12 hours.

What is the significance of tight junctions in epithelial cells?

Tight junctions are crucial for maintaining the integrity of the intestinal barrier and regulating permeability.

How often should the culture medium be refreshed?

The culture medium should be refreshed every two to three days.

What microscopy technique is used to observe the cells?

Phase-contrast microscopy is used to observe the cell cultures.

What is the purpose of using collagen-coated inserts?

Collagen-coated inserts provide a suitable surface for cell adhesion and growth.

Generation of a Human Colonoid Monolayer with Barrier Function to Study Host–Pathogen Interactions

JoVE Encyclopedia of Experiments

Microbiology

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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.