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

1. Generation of spheroids

1. Prepare and autoclave 2% agarose in 1x phosphate-buffered saline (PBS) (e.g., 1 g agarose in 50 mL of 1x PBS) and autoclave 35- and 81-microwell rubber molds.
   CAUTION: Avoid using low-melting agarose for generating the agarose casts for immunohistochemistry (IHC) processing.
2. Prepare agarose casts
   NOTE: 35-microwell casts are used for invasion, immunofluorescence (IF) and cell isolation assays. 81-microwell casts are used for cytotoxicity, IHC, cell isolation and ribonucleic acid (RNA) extraction assays.
   1. After the agarose has been autoclaved, let molten agarose cool to about 60‒70 °C. In a cell culture hood, use aseptic technique and pipette 500 µL of molten agarose into an 81-microwell rubber mold per well or 330 µL into a 35-microwell rubber mold per well.
      CAUTION: Avoid creating bubbles while mixing or pipetting agarose. Remove any bubbles by gently pipetting.
   2. After the agarose is solidified, carefully flex the rubber mold to remove the agarose cast from it. Hereby, place hands above the appropriate well-plate and let the agarose cast drop right into one well of the well-plate.
      NOTE: Flex the rubber mold at different positions in order to avoid over flexing. It might be helpful to flex the rubber mold and push gently from the bottom at the same time.
   3. To equilibrate the agarose casts, add cell culture medium, consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (2.5 mL/well for 12-well plate and 1 mL/well for 24-well plate). Put the well-plate into a cell culture incubator (37 °C, 5% CO₂) and incubate for 1 h.
3. Meanwhile, prepare cell culture for seeding.
   NOTE: The total cell seeding number per agarose cast is to be decided by the investigator. For murine primary pancreatic cancer cell lines, 35,000 cells/35-microwell cast and 81,000 cells/81-microwell cast (in average 1000 cells/spheroids) are seeded for all following assays. The average diameter of a spheroid is about 150‒200 µm after 48 h.
4. Remove cell culture medium surrounding the agarose cast with a P1000 pipette first by tilting the well-plate. Then carefully remove the medium in the seeding chamber.
5. Carefully seed the prepared tumor cell suspension drop-wise into the cell seeding chamber. Carefully put the well-plate back into the cell culture incubator (37 °C, 5% CO₂) for 15 min.
   NOTE: During this step the cells will settle into the microwells of the agarose cast.
6. Add additional medium to the outside of the agarose cast (2.5 mL/well for 12-well plate and 1 mL/well for 24-well plate).
7. Put the well-plate back into the cell culture incubator (37 °C, 5% CO₂) for 48 h.
   NOTE: Usually it takes up to a few hours for the cells to form spheroids in the microwells. In general, solid tumor cell spheroids are formed after 24 h and are stable after 48 h. However, this can vary between different cell lines. If necessary, change cell culture medium by carefully tilting the well-plate with the agarose casts and removing the surrounding cell culture medium with a P1000 pipette. Then carefully add fresh medium by pipetting it along the wall of the well. Usually it is not necessary to change the media within the casts due to its small volume and the risk of removing the spheroids.

2. Co-culture with T-cells

1. Resuspend the required number of T-cells in 75 µL (35-microwell cast) or in 190 µL (81-microwell cast) of the appropriate T-cell culture medium (RPMI supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin).
2. Remove the cell culture medium surrounding the agarose cast with a P1000 pipette first by tilting the well-plate. Then slowly and carefully remove the medium within the cast by gently targeting one corner of the seeding chamber with a P200 pipette while keeping the well-plate tilted with the other hand.
3. (Critical step 1) As there is a risk of removing spheroids, control for loss of spheroids by comparing the number of spheroids within the microwells before and after removing the medium by viewing under the microscope at 10x magnification.
4. Carefully seed the T-cell suspension drop-wise into the seeding chamber of the agarose cast by holding the P200 pipette about 0.5 cm above the cast.
5. (Critical step 2) There is a risk of flushing out the spheroids while adding the T-cells. Therefore, it is critical to add the T-cells very slowly and about 0.5 cm above the seeding chamber.
   NOTE: One possibility to decrease the number of flushed out spheroids is by pointing the pipette to one corner of the seeding chamber while adding the T-cells, thus only risking the spheroids in this corner to be flushed out.
6. Carefully place the well-plate back into the cell culture incubator (37 °C, 5% CO₂) for 15 min.
7. Take the well-plate out from the incubator and add fresh cell culture medium (Roswell Park Memorial Institute media (RPMI) supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin) around the agarose cast by slowly pipetting it along the wall of the well.
8. Put the well-plate back into the cell culture incubator for 48 h.