Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture
Published: April 30, 2023
Abstract
Source: Slone, W. L., et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes a method for spatial separation of three leukemic cell subpopulations: the suspended leukemic cells freely floating in the media, phase bright leukemic cells adhered to the surface of the mesenchymal stromal cell monolayer, and the phase dim leukemic cells that have migrated beneath the mesenchymal stromal cell monolayer from 2D co-culture.
Protocol
1. Separating 3 Subpopulations within Co-culture
- Collection of suspension (S) tumor subpopulation.
- 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.
- Collection of Phase Bright (PB) tumor subpopulation.
- 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.
- Aspirate with pipette and collect media in a 15 ml conical tube. The collected cells are the PB subpopulation.
- Collection of Phase Dim (PD) tumor subpopulation.
- 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.
- Remove plate out of incubator and gently tap sides of the plate to dislodge adherent BMSC/OB.
- Add 1 ml fetal bovine serum (FBS) and pipette up and down 3-5 times to break apart large cell aggregates.
- Collect media with cells in a 15 ml conical tube. These cells are the unpurified PD subpopulation with BMSC/OB as well.
- 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.
Divulgations
The authors have nothing to disclose.
Materials
| 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 prepation |
| Cell lines | |||
| Human Osteoblasts | PromoCell | C-12720 | Human osteoblast were cultured according to the supplier’s recommendations. |
| Human Bone Morrow 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. |
Citer Cet Article
Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture. J. Vis. Exp. (Pending Publication), e20261, doi: (2023).