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 follow