Proximal Culture System: An In Vitro Culture Technique to Study Paracrine Signaling Between Cells

Begin by detaching the ovarian cancer cells from an 80% confluent mixture with 1 milliliter of 0.25% trypsin for 40 to 60 seconds, followed by neutralization of the reaction with 6 milliliters of complete growth medium. Collect the cells by centrifugation and resuspend the pellet in 5 milliliters of fresh complete growth medium.

After counting, dilute the cells to a 100,000 ovarian cancer cells per milliliter of complete growth medium concentration, and place three inverted 0.4-micrometer pore tissue-culture-treated polycarbonate membrane cell culture inserts per experimental condition in an appropriately sized sterile tissue culture dish. Label the inserts according to the experimental condition, and carefully pipet the cancer cells onto the bottom of each insert in concentric circles, starting from the middle of the membrane and moving outward, to form an 800 microliter dome.

Be extremely careful when seeding the cells onto the bottom surface of the insert so that the dome of the medium-containing cells does not run off the edges of the insert during the incubation.

When all of the inserts have been seeded, cover the dish and carefully place the inserts into the cell culture incubator. After about 4 hours, detach the HPMC with 2 milliliters of 0.25% trypsin for one to two minutes, followed by neutralization of the reaction with 12 milliliters of complete growth medium.

Collect the HPMC by centrifugation and resuspend the pellet in 10 milliliters of complete growth medium for counting. Dilute the cells to a 300,000 HPMC per milliliter of complete growth medium and add 2.5 milliliters of fresh complete growth medium to each well of a 6-well culture plate.

Using sterile forceps, place each insert right side up into each well of the 6-well plate so that the lower ovarian cancer cell attached surfaces are immersed in the media. Then, seed 1.5 milliliters of HPMC into the well of each insert and place the plate in the cell culture incubator for 72 hours.