A Three-Dimensional Co-culture Model of Infiltrating T Cells in Tumor Spheroids

Begin with a culture plate containing an agarose cast. The cast comprises a seeding chamber with multiple microwells.

Each microwell contains a tumor spheroid in a culture medium. The spheroids are three-dimensional aggregates of tumor cells.

First, remove the medium surrounding the agarose cast. Then, remove the medium within the cast from one corner of the seeding chamber to avoid the loss of spheroids.

Introduce a suspension of tumor-specific T cells dropwise into the seeding chamber, allowing the cells to settle into the microwells containing the spheroids.

Next, add a culture medium around the agarose cast and incubate the plate under physiological conditions to develop a co-culture.

During incubation, the T cells surround the spheroid. A subset of T cells infiltrate the spheroid, while others remain in the periphery, establishing a three-dimensional co-culture model.

To set up a T cell co-culture, resuspend the appropriate number of T cells in 190 microliters of an appropriate T cell culture medium per well, and tilt the 12-well plate to allow removal of the cell culture medium surrounding the agarose cast first, then in the seeding chamber, without removing the spheroids.

Use a light microscope to check whether any spheroids have been aspirated, and holding the P200 loaded pipette about 1/2 a centimeter above each cast, carefully seed the T cells onto the casts in a dropwise fashion, without dislodging the spheroids. When all of the casts have been seeded, carefully return the plate to the cell culture incubator for 15 minutes, before adding fresh cell culture medium supplemented with fetal bovine serum to the outside of each cast for another 48-hour incubation.