The overall goal of this procedure, is to better model resistant acute lymphoblastic leukemia, or ALL, in vitro. This method can help answer key questions in the treatment of refractory ALL about how the bone marrow microenvironment influences ALL cell chemotherapeutic resistance. The main advantages of this technique are that it is considerably cheaper and less time consuming than utilizing traditional animal models to examine drug advocacy on refractory ALL cells.
Begin by seeding five to 20 times 10 to the sixth leukemic cells in 10 milliliters of tumor-specific culture medium onto a 100 millimeter plate of 80 to 90%confluent bone marrow stromal cells or osteoblasts. Every fourth day, tilt the plate to the side and aspirate all but one milliliter of the medium, taking care not to disturb the adherent cell layer. Then, carefully add nine milliliters of fresh leukemic cell culture medium dropwise into the corner of the plate along the side to ensure a minimal disruption of the adherent cells.
After the 12th day of co-culture, gently pipette the culture medium up and down over the plate approximately five to 10 times to collect the floating leukemic cells. And transfer the cells into a 15 milliliter conical tube. Then, reseed the cells onto a new plate of 80 to 90%confluent bone marrow stromal cells, or osteoblasts, as just demonstrated.
By day four, three subpopulations of leukemic cells will have formed, with the suspended leukemic cells freely floating in the medium. The Phase Bright leukemic cells adhere to the surface of the adherent cell monolayer, and the Phase Dim leukemic cells migrated beneath the monolayer. To prepare the G10 bead columns, first rewarm 30 milliliters of cell culture medium per column to 37 degrees Celsius in a water bath.
Next, use tweezers to pull glass wool into thin, loose strands. And add multiple layers of lightly packed strands to one 10 milliliter syringe per column needed. When the syringe is 2/3 full, attach a one-way stopcock in the closed position to the tip, and clamp the syringe onto a ring stand high enough that a 50 milliliter conical collection tube can be placed beneath the stopcock.
Now, place a collection tube under the syringe column. And use a 10 milliliter pipette to add drops of G10 particles, suspended in PBS, on top of the glass wool. Continue adding the G10 particles until a two milliliter pellet forms.
Then, add two milliliter aliquots of the pre-warmed medium to the column. And slowly open the stopcock valve, so that the medium flows out of the column dropwise. When 10 milliliters of medium have fully drained through the column, close the stopcock.
Discard the flow-through, and place a new collection tube under the column. To harvest the suspended tumor subpopulation, aspirate the medium from the leukemic cell co-culture plate, and use the same medium to gently rinse the plate one time. Then, transfer the floating suspension leukemic cell subpopulation into a 15 milliliter conical tube.
To harvest the Phase Bright tumor subpopulation, add 10 milliliters of fresh medium back onto the co-culture plate. And rinse approximately five times vigorously enough to remove the adherent leukemic cells without dislodging the adherent cell monolayer. Then, transfer the Phase Bright subpopulation into its own 15 milliliter conical tube.
To harvest the Phase Dim tumor subpopulation, remove the remaining medium with a one milliliter PBS rinse. And detach the cells with three milliliters of trypsin at 37 degrees Celsius. After five minutes, gently tap the sides of the plate to dislodge the adherent cells, followed by the addition of one milliliter of FBS to stop the enzymatic reaction.
Then, rinse the serum three to five times to break apart any large cell aggregates. And transfer the unpurified Phase Dim subpopulation into a new 15 milliliter conical tube. When all of the subpopulations have been collected, spin down the cells.
And re-suspend each of the pellets in one milliliter of pre-warmed medium. With the stopcock completely closed, for each subpopulation, use a 1, 000 microliter pipette to add the entire one milliliter volume of cells, dropwise, onto the top of one of the prepared G10 columns, making sure the cells remain on top of, or within, the G10 pellet. Allow the cells to incubate on the G10 pellet for 20 minutes at room temperature.
Then, add one to three milliliters of fresh pre-warmed medium to the column. And open the stopcock so that the medium slowly exits the columns, dropwise. Continue to add pre-warmed medium to the columns, in one to two milliliter increments.
When a total of 15 to 20 milliliters of leukemic cells has been collected, close the stopcock and cap the collection tube. Then, spin down the cells and re-suspend the pellet in the appropriate downstream analysis buffer. Following trypsinization of the adherent cell layer, two distinct populations of cells are observed by forward versus side scatter analysis.
After G10 separation, a pure population of ALL cells is recovered. Of particular interest, all of the Phase Dim cells recovered from bone marrow stromal cell, or osteoblast co-cultures, exhibit little to no death following their exposure to cytotoxic chemotherapy. While attempting these procedures, it's important to remember to perform these steps as properly as possible to reduce any phenotypic changes that may occur in the leukemic cells.
This technique provides a way for researchers in the field of leukemia research to model the chemotherapy-resistant leukemia in an in vitro model that more closely resembles the bone marrow microenvironment.
