This video describes the separation of acute lymphoblastic leukemia cells from bone marrow stromal cells using gel-type ten cross-linked dextran G-10 columns. This method helps recover the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells generating a "phase dim" tumor cell population.
Protocol
1. Loading Co-culture Cells onto G10 Column
NOTE: Make sure stopcock is completely closed before adding media containing cells to G10 column. Also, each subpopulation must be ran over a separate G10 column so not to introduce any bias between populations in downstream analysis.
Using a 1,000 µl pipette, add 1 ml of each cell subpopulation in pre-warmed media to a separate G10 column drop-wise. Ensure that the media containing the cells remains on top or within G10 pellet. Allow cells to incubate on G10 pellet for 20 min at RT. NOTE: Stopcock remains closed for duration of incubation.
2. Collecting Leukemic Cells from G10 Column
Add 1-3 ml pre-warmed media to each G10 column. Open stopcock valve and allow media to slowly exit the column drop-wise. NOTE: It is crucial to maintain a slow flow rate from the column or the G10 pellet containing BMSC/OB can wash out of the column and contaminate the isolated leukemic cells.
Continue to add pre-warmed media in small increments (1-2 ml) to G10 column until a total of 15 to 20 ml has run through column and has been collected. Close stopcock valve and cap collection tube. NOTE: If a G10 particle pellet is seen at the bottom of collection tube, gently remove media from the tube leaving G10 particle pellet undisturbed and transfer to new tube.
Centrifuge collected media at 400 x g for 7 min at RT. Remove supernatant and resuspend cell pellet in buffer appropriate for downstream application.
Cells are now a pure population of leukemic cells free of BMSC or OB contamination and are ready to be applied to downstream applications at user discretion. NOTE: Leukemic cell viability should remain unchanged when passing cells through G10 columns.
Divulgazioni
The authors have nothing to disclose.
Materials
G10 sephadex beads
Sigma
G10120
Referred to in manuscript as gel type 10 cross-linked dextran particles
10 ml sterile syringe
BD
309604
1-way stopcocks
World Precision Instruments, Inc.
14054-10
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 (*)
0.05% Trypsin
Mediatech, Inc.
25-053-CI
REH
ATCC
ATCC-CRL-8286
REH cells were cultured according to the supplier’s recommendations and recommended media
Glass wool
Pyrex
3950
50 ml conical centrifuge tubes
World Wide Medical Products
41021039
Used as collection tubes
15 ml conical centrifuge tubes
World Wide Medical Products
41021037
Used for cell collection
Fetal Bovine Serum
Sigma
F6178
SD-1
DSMZ
ACC 366
SD-1 were cultured according to the supplier’s recommendations and recommended media
G-10 Column Based Leukemia Cell Sorting: A Method to Purify Acute Lymphoblastic Leukemia Cells from Bone Marrow Stromal Cells. J. Vis. Exp. (Pending Publication), e20262, doi: (2023).