An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

The overall goal of this procedure is to quantify the chemo sensitivity of multiple myeloma primary cells to a panel of chemotherapeutic agents. This is accomplished by first mixing multiple myeloma cells isolated from fresh bone marrow aspirate with pre culture, patient derived stroma and collagen. In the second step, the CEL mix is seeded onto a multi-well plate.

The cells are then treated with the chemotherapeutic agents of interest, and the plate is transferred to the incubator of a motorized stage microscope for sequential imaging of the cells over a 96 hour incubation period. Ultimately, a digital image analysis algorithm can be used to generate viability curves for each drug and concentration. The main advantages of this technique over existing endpoint viability assays are that it can be used to test several chemotherapeutic agents versus a single patient specimen, and that the viability of the patient cells can be measured longitudinally over several days without needing to separate the cells from the stroma or the matrix.

The implications of this technique extend toward the therapy of hematopoietic tumors as it can theoretically be used to predict clinical response to specific chemotherapy regimens. We first had the idea for this protocol when we realized that replacing our original microfluidic slides with multi-well plates would greatly reduce the cost and time required to complete the experiment. To prepare primary multiple myeloma, bone marrow derived mesenchymal stem cell co cultures with a multi-channel pipetter begin by suspending the cells at six times the final concentration in RPMI 1640.

Medium next, mix 50 microliters of each cell type with freshly prepared type one collagen to a final volume of 300 microliters. Then use a multi-channel pipette to manually dispense eight microliters of the cell matrix mix into the center of each well of a 3 84. Well plate and centrifuge the plate to concentrate all of the cells to the same focal plane.

After spinning, incubate the plates under cell culture conditions for one hour to allow the gel to polymerize. Then add 80 microliters of supplemented growth medium to each well and to incubate the plate overnight to allow the stroma to adhere to the bottom of the plate. To prepare multiple myeloma stroma co cultures with a robotic pipetter, begin by suspending 300 microliters of CD 1 38 positive multiple myeloma cells and 300 microliters of stromal cells in RPMI 1640, medium at six times the final desired density as just demonstrated.

Then mix both cell types together in a 1.5 milliliter tube labeled CD 1 38 positive and placed the tube in a cell culture incubator. Next, suspend 60 microliters of the appropriate multiple myeloma cell line and 60 microliters of the stromal cells in RPMI 1640, medium at six times the final desire density and to mix both cell types together in a 1.5 milliliter tube labeled this second tube cell line and incubated at 37 degrees Celsius as well. On the robotic pipetter load the cell seeding script file.

Then place a 200 microliter pipette tip box into station A, a microtiter plate into station B and a sterile 3 84. Well plate into station E of the robot. Next, mix the CD 1 38 positive cell mixture with the tube of freshly prepared CD 1 38 premixed medium, and transfer 1.5 milliliters of the resulting cell solution to the number one well of the microtiter plate.

Place the rest of the cells on ice and start the program. The robot will seed the first 176 wells of the 384 well plate and then pause. Use this time to transfer the remaining CD 1 38 positive cells to well number two of the microtiter plate and resume the program.

The robot will see the remaining 144 wells of the plate and pause again. Now mix the cells from the cell line tube with a tube of freshly prepared cell line premix and transfer the contents to well number three of the microtiter plate. Then resume the program again, allowing the robot to seed the remaining 64 wells of the 3 84 well plate.

When the last well has been seeded, spin down the cells and transfer the co cultures to the cell culture incubator for collagen polymerization. After one hour, load the media layer script on the robot and place a 120 microliter pipette tip box in station A, a reagent reservoir with 31 milliliters of RPMI 1640 medium supplemented with fetal bovine serum patient plasma and antibiotics in station B and the CED 3 84 well plate into station E.Then run the program. The robot will transfer 81 microliters of the supplemented medium to each well.

When the robot is finished, return the cells to the incubator to allow the stroma to adhere to the substrate overnight. To treat the seeded cells with the drug of interest, first load the media layer drug plate script in the robotic pipetter user interface. Then place a 120 microliter pipette tip box in station A, a reservoir with 21 milliliters of RPMI 1640 supplemented with FPS patient plasma and antibiotics in station B and an empty 3 84 well plate in station E run the program, the robotic pipetter will add 30 microliters of medium to all of the wells.

The 3 84 well plate. Then following the template at 200 microliters of drug at 20 times the maximum concentration to each well of a microtiter plate to the control. Well add medium supplemented with FPS patient plasma and antibiotics.

Then place a 120 microliter pipette tip box in station A, the microtiter plate of drugs in station B and A 3 84, well plate of medium in station C load the drug plate program. The robotic pipetter will create a serial one to three dilution as illustrated in this figure. Now place a fresh 120 microliter pipette tip box in station A, the 3 84 well plate of diluted drug in station B and the 3 84 well plate of seeded cells into station C and run the drug add program.

The robotic pipetter will transfer eight microliters of drug from each well of the drug plate to its counterpart in the cell plate. After the last well has been treated, immediately transfer the cell plate to the incubator of the digital microscope. Then to image the cells, clean the interior of the benchtop incubator with an ethanol wet wipe, and carefully switch the lid of the plate with the lid of the incubator.

Next, follow the software steps for adding the landmarks for the regions of interest to be imaged successively during the experiment. Finally, using a five or 10 x magnification objective, bring the cells into focus until the multiple myeloma cells appear as bright discs surrounded by a dark ring and the stromal cells are barely visible. In this experiment, the live multiple myeloma cells were pseudo colored in green with little to no green signal observed in the stromal and human umbilical vein endothelial cells at the end of the experiment at the highest drug concentration when all of the multiple myeloma cells were dead.

The robot software was then used to tart the gradual loss of viability resulting from the exposure to the different drug concentrations over time. It is important to carefully observe all of the seeded wells before starting the imaging process as artifacts may mislead the image analysis software and lead to false results. For example, here the result of an excessive density of stromal cells or too much time between the trypsin, ization and seeding is shown resulting in the formation of cell clumping as seen in this image.

If the cell lines are seeded at two high densities, they will form colonies as they replicate reducing the accuracy of the digital image analysis algorithm as it becomes increasingly difficult to discern one cell from the other here too few multiple myeloma cells were seeded when the number of multiple myeloma cells obtained from the bone marrow aspirate is below 500, 000. It is necessary to determine the dead volume for the tube being used before seeding the cells and to include this volume in the dilution calculations. Once mastered, this technique can be performed in two to three hours.

When attempting this procedure, it's important to remember to keep the premixed medium containing the collagen on ice to prevent polymerization before the cells are ready. It also helped in exploring how the heterogeneity of chemo sensitivity of the tumor burden can determine the depth and duration of the patient response.