Cells were inoculated into filter membrane Petri dishes and cultured in excess culture solution in multiwell culture plates.

Operation method

Petri dish

Principle

Cells were inoculated into filter membrane Petri dishes and incubated in excess culture solution in multiwell culture plates.

Materials and Instruments

Approx. 0.5 x 1,000,000 cells per square centimeter of filter membrane
Growth medium
Petri dish, porous plate, curved forceps, dropper.

Move

1. Place the membrane petri dish into a petri plate or petri dish.

2. Add the culture solution and tilt the petri dish so that the solution fills the space underneath the membrane, displacing as much air as possible. Add culture solution to the level of the membrane (2.5 ml per well for 6-well plates; 1.0 ml per well for 24-well plates).

3. Place the plate horizontally and add 2 ml of culture solution containing ^2x106 cells to a 25 mm diameter membrane, and 200 ul of culture solution containing ^5x105 cells to a 6.5 mm diameter membrane, being careful not to puncture the membrane.

4. Place the plate in a protective box and place it in a humidified container. The plate is placed in a protective box and incubated in a humidified _CO2 incubator. It is critical to avoid shaking the box and the culture should not be moved around in the incubator to avoid spillage and contamination.

5. Monolayers can be formed in 3-5 d. Histotypic differentiation (e.g., polarity transfer) may take 5-10 d or longer to establish.

6. Cultures can be maintained indefinitely, with culture medium changed or nested sets transferred to new wells or dishes every 3-5 d. The culture medium can be changed every 3-5 d or with a new well or dish transferred to a new well or dish.

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