G-10 Column Based Leukemia Cell Sorting: A Method to Purify Acute Lymphoblastic Leukemia Cells from Bone Marrow Stromal Cells

Overview

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.

1. 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

1. 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.
2. 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.
3. Centrifuge collected media at 400 x g for 7 min at RT. Remove supernatant and resuspend cell pellet in buffer appropriate for downstream application.
4. 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.

Materials

Name	Company	Catalog Number	Comments
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
100 x 20 mm Cell Culture Dishes	Greiner Bio-One	664160