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 human bone marrow stromal cells.

– Begin by seeding 5 to 20 times 10 to the sixth leukemic cells in 10 milliliters of tumor specific culture medium onto a 100 millimeter plate of 80% to 90% confluent bone marrow stromal cells or osteoblasts. Every fourth day, tilt the plate to the side and aspirate all but 1 milliliter of the medium, taking care not to disturb the adherent cell layer. Then, carefully add 9 milliliters of fresh leukemic cell culture medium drop wise into the corner of the plate along the side to ensure a minimal disruption of the adherent cells.

After the 12th day of co-culture, gently pipette the culture medium up and down over the plate approximately 5 to 10 times to collect the floating leukemic cells, and transfer the cells into a 15 milliliter conical tube. Then, reseed the cells onto a new plate of 80% to 90% confluent bone marrow stromal cells or osteoblasts, as just demonstrated. By day 4, three sub populations of leukemic cells will have formed, with the suspended leukemic cells freely floating in the medium, the phase-bright leukemic cells adhered to the surface of the adherent cell monolayer, and the phase-dim leukemic cells migrated beneath the monolayer.