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

Protocol

1. Separating 3 Subpopulations within Co-culture

1. Collection of suspension (S) tumor subpopulation.
   1. Aspirate media from co-culture plate with pipette and gently re-apply the same media to rinse the plate and collect media containing leukemic cells in a 15 ml conical tube. The leukemic cells collected are the S subpopulation.
2. Collection of Phase Bright (PB) tumor subpopulation.
   1. Add 10 ml fresh media back onto co-culture plate. Rinse vigorously by pipetting added media up and down approximately 5 times to remove adherent leukemic cells but not hard enough to dislodge adherent BMSC/OB component.
   2. Aspirate with pipette and collect media in a 15 ml conical tube. The collected cells are the PB subpopulation.
3. Collection of Phase Dim (PD) tumor subpopulation.
   1. Rinse plate with 1 ml PBS to remove remaining media. Trypsinize co-culture plate with 3 ml trypsin and place into 37 °C incubator for 5 min.
   2. Remove plate out of incubator and gently tap sides of the plate to dislodge adherent BMSC/OB.
   3. Add 1 ml fetal bovine serum (FBS) and pipette up and down 3-5 times to break apart large cell aggregates.
   4. Collect media with cells in a 15 ml conical tube. These cells are the unpurified PD subpopulation with BMSC/OB as well.
4. Centrifuge 3 isolated subpopulations at 400 x g for 7 min. Aspirate and discard supernatant then individually resuspend pellets in 1 ml pre-warmed media. Cells are ready to be loaded onto a G10 column.

Materials

Name	Company	Catalog Number	Comments
50 ml conical centrifuge tubes	World Wide Medical Products	41021039	Preservative solution for cytology specimens
15 ml conical centrifuge tubes	Novacyt	NA	Used for cell collection
Fetal Bovine Serum	Sigma	F6178
0.05% Trypsin	Mediatech, Inc	5229 TEC 5	25-053-CI
100 x 20 mm Cell Culture Dishes	Greiner Bio-One	664160
Culture media
Osteoblast culture media	PromoCell	C-27001	For human osteoblast media
RPMI 1640 media	Mediatech, Inc.	15-040	For tumor media preparation
Cell lines
Human Osteoblasts	PromoCell	C-12720	Human osteoblast were cultured according  to the supplier’s recommendations.
Human Bone Marrow Stromal Cells	WVU Biospecimen Core		De-identified primary human leukemia and bone marrow stromal cells (BMSC) were provided by the Mary Babb Randolph Cancer Center (MBRCC) Biospecimen Processing Core and the West Virginia University Department of Pathology Tissue Bank. BMSC cultures were established as previously described (*)
Leukemic cells
REH	ATCC	ATCC-CRL-8286	REH cells were cultured according to the supplier’s recommendations and recommended media.
SD-1	DSMZ	ACC 366	SD-1 were cultured according to the supplier’s recommendations and recommended media.