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

1. Advanced Preparation

1. Culturing bone marrow stromal cells (BMSC) and osteoblasts (OB).
   1. Maintain both BMSC or OB at 37 °C in 6% CO₂ and grown on 10 cm tissue culture plates until 90% confluency is reached.
   2. Trypsinize BMSC or OB cells and split 1:2 onto new 10 cm plates. The cells are grown to these standards until needed for leukemic co-culturing.

2. Establishing and Maintaining Co-culture

1. Add 5-20 x 10⁶ leukemic cells in 10 ml of tumor specific culture media onto an 80%-90% confluent BMSC or OB plate.
   NOTE: Our lab maintains co-cultures at 37 °C in 5% O₂ to better recapitulate the bone marrow microenvironment which has been shown to range from 1% to 7%. However, maintaining co-cultures at this oxygen tension is not critical for the establishment of the three leukemic subpopulations and is at the discretion of the lab.
2. Every 4^th day remove all but 1 ml of media (including leukemic cells in suspension) and replace with 9 ml fresh leukemic culture media. When removing 9 ml of media from plate, be careful not to disturb the bone marrow stromal cells (BMSC) or osteoblast (OB) adherent layer.
   1. Remove media by tilting plate to the side and aspirate media in the corner of the plate. Additionally, when adding fresh media, be sure to add drop wise in the corner of the plate against the sidewall to ensure minimal disruption of the BMSC or OB adherent layer.
3. After the 12^th day of co-culture, rinse leukemic cells from BMSC or OB layer by pipetting culture media from dish up and down gently over the dish approximately 5 to 10 times and then collect in 15 ml conical tube. Reseed onto new 80%-90% confluent BMSC or OB plate as described in step 2.1.
   NOTE: The gentle rinsing of the co-culture as described in step 2.3 will remove suspended and phase bright leukemic cells without disrupting the BMSC or OB monolayer. This allows only tumor cells to be transferred to the next co-culture plate. This 12 day cycle can be repeated as many times as needed based on user needs.