Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

- When leukemic cells are co-cultured with mesenchymal stromal cells, they interact with stromal cells and form three subpopulations. Freely floating suspended cells, phase-bright cells adherent to the surface of the mesenchymal monolayer, and phase-dim cells beneath the mesenchymal monolayer. To separate each subpopulation, take a tissue culture plate with a mesenchymal stromal cell monolayer.

Seed leukemic cells present in tumor-specific culture media onto the monolayer, and incubate the plate for the desired duration. Next, collect tumor cells in suspension by pipetting the freely floating cells into a separate tube. Now, add the fresh media to the plate and pipette vigorously to dislodge the phase-bright tumor cells from the mesenchymal monolayer surface.

Collect these cells by pipetting them in a separate tube. Add trypsin to the plate for removing the adherent mesenchymal cells. Add fetal bovine serum, or FBS, to stop the action of trypsin and to break apart the cell aggregates. Now, collect the phase-dim cells in a tube. Centrifuge all three tubes containing different cell subpopulations individually and resuspend the pellet in fresh media for further analysis. In the following protocol, we will isolate suspension, phase-bright, and phase-dim leukemic cells from 2D co-culture.

- To harvest the suspended tumor subpopulation, aspirate the medium from the leukemic cell co-culture plate and use the same medium to gently rinse the plate one time. Then, transfer the floating suspension leukemic cells subpopulation into a 15 milliliter conical tube. To harvest the phase-bright tumor subpopulation, add 10 milliliters of fresh medium back onto the co-culture plate

and rinse approximately five times vigorously enough, to remove the adherent leukemic cells without dislodging the adherent cell monolayer. Then, transfer the phase-bright subpopulation into its own 15 milliliter conical tube. To harvest the phase-dim tumor subpopulation, remove the remaining medium with a one milliliter PBS rinse and detach the cells with three milliliters of trypsin at 37 degrees Celsius.

After five minutes, gently tap the sides of the plate to dislodge the adherent cells, followed by the addition of one milliliter of FBS to stop the enzymatic reaction. Then, rinse the serum three to five times to break apart any large cell aggregates and transfer the unpurified phase-dim subpopulation into a new 15 milliliter conical tube. When all of the subpopulations have been collected, spin down the cells and resuspend each of the pellets in one milliliter of pre-warmed medium.