Mesothelial Clearance Assay: An In Vitro Technique to Quantify the Invasion Ability of Ovarian Cancer Spheroids into Mesothelial Cell Monolayers

To begin this procedure, pre-coat the wells of a 6-well glass-bottom MatTek dish with fibronectin. Add 2 milliliters of fibronectin/PBS solution to each well of the dish and incubate at room temperature for 30 minutes. The green fluorescent protein-expressing mesothelial cells, for forming a mesothelial cell monolayer, are cultured in 10% base medium. Trypsinize a plate of mesothelial cells and spin down in a tabletop centrifuge at 900 RPM for three minutes.

Aspirate the supernatant and resuspend cells in 10% base medium. Adjust to the desired concentration with 10% base medium. When the 30-minute fibronectin incubation of the MatTek dish is complete, wash the wells with 2 milliliters of PBS. Aspirate the PBS and plate 6 times 10 to the fifth mesothelial cells per well in each well of the 6-well MatTek dish. Incubate the MatTek dish in a 37 degrees Celsius cell culture incubator overnight to allow the mesothelial cells to attach to the dish and form a monolayer.

Use a pipette to collect the ovarian cancer spheroids from the 96-well poly-HEMA coated plate. Aspirate the medium from one well of the 6-well MatTek dish containing a mesothelial cell monolayer. Wash once with 2 milliliters of PBS. Add all of the spheroids from the 96-well plate to one well of the MatTek dish. This is approximately three times the number of spheroids that are going to be imaged to account for spheroids landing on the part of the dish that cannot be imaged.

Since we want to image only one spheroid per field of view, aggregation of the spheroids must be avoided. Rock the dish gently from side to side to distribute the spheroids evenly across the monolayer before they attach.

Place the MatTek dish on the stage of an inverted wide-field fluorescence microscope capable of performing time-lapse imaging for the duration of at least eight hours. Use a motorized stage to image multiple positions in the dish, with multiple spheroid intercalation events, in a single experiment. The ovarian cancer cell spheroids will settle to the bottom of the dish and attach to the mesothelial cell monolayer. Collect GFP, RFP, and phase images of 20 plus spheroid/monolayer interactions every ten minutes for eight hours.