2D Co-culture of Leukemic Cells with BMSCs: A Method to Obtain Subpopulations of Leukemic Cells

- In a 2D co-culture system, multiple cell types are grown together in flat culture plates. This culture system facilitates the study of cell interactions and behavior in vitro. To establish leukemic cell co-culture with Bone Marrow Stromal Cells, or BMSCs, add leukemic cell suspension to an adherent monolayer culture of BMSCs. Incubate the plate under appropriate culture conditions. Maintain the co-culture by replacing 90% of the culture medium on every fourth day.

During this 2D co-culture, the leukemic cells interact with the BMSCs and localize to distinct regions in the culture, forming three sub populations. Phase contrast microscopy allows differentiating the three sub populations visually. The first group of leukemic cells that appears freely floating in the medium are categorized as suspended cells. The second group of leukemic cells that have adhered to the surface of the BMSC monolayer appears bright and are categorized as phase-bright cells.

The third group of leukemic cells that have migrated beneath the monolayer appears dim and are categorized as phase-dim cells. In the following protocol, we perform the co-culture of Acute Lymphoblastic Leukemia, or ALL cells, with h