Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

The overall goal of this procedure is to form cancer cell pH in a standard high throughput format in order to use them in anti-cancer drug testing. This is accomplished by first dissolving two polymers, polyethylene glycol, and dextran in cell culture medium at predetermined concentrations to form a successful aqueous two-phase system. The second step is to prepare the cells of interest at twice the desired cell density and mix the cell suspension with an equal volume of the aqueous dextran phase.

Then fill a 96 well plate with the aqueous polyethylene glycol phase and dispense sub microliter Droplets of cell containing dextran phase drops into the wells using a liquid handling robot. The next step is to confirm spheroid formation in the 96 well plates after 24 hours of incubation and to directly add anti-cancer drugs of interest to the wells. Finally, a standard plate reader compatible viability assay is used to show the percent viability of drug treated steroids normalized against control non-treated spheres.

The main advantage of this technique over existing methods like the hanging drop method or the micro fabricated well approach is that it does not require customized plates or devices and allows high throughput sphere formation and drug testing. This method can help answer key questions in cancer research and expedite discovery of anti-cancer chemotherapeutics. The implications of this technique extend towards the therapy of cancer because it allows high throughput screening of chemical compounds with more relevant tumor models to identify new anti-cancer drugs.

Individuals new to this method will conveniently form cancer cell steroids and micro well plates and will easily conduct drug treatment experiments using standard lab wear and robotic tools. To begin, weigh out 0.5 grams of polyethylene glycol and add it to 9.5 milliliters of complete growth medium in a sterile 15 milliliter conical tube Vortex, the mixture for one minute to prepare 10 milliliters of a 5%weight per volume aqueous polyethylene glycol phase. Then weigh out 0.128 grams of dextran and add it to 0.87.

Two milliliters of complete growth medium in a sterile 1.5 milliliter micro centrifuge tube Vortex. This mixture for one minute to prepare one milliliter of a 12.8%weight per volume aqueous dextran phase. Place both the polyethylene glycol and dextran solutions in a 37 degree Celsius water bath for one hour.

To assist with complete dissolution. Ensure that the caps stay above water level to avoid possible contamination after one hour. Remove the polyethylene glycol face solution and load it into a five milliliter sterile plastic syringe.

Pass the solution through a 0.2 micron pore syringe top filter to remove any impurities. Store both the polyethylene glycol and dextran phase solutions at four degrees Celsius for up to 24 hours before use. Grow cancer cells of interest such as the M-D-A-M-B 1 57 breast cancer cell line until they are 90 to 100%confluent.

Then harvest the cells using standard techniques and count the yield on a hemo cytometer resuspend the pellet to a concentration of 50 million cells per milliliter in a mixture of half growth, medium and half strand solution. This ensures a density of about 15, 000 cells per 0.3 microliter droplet dispense 50 microliters of the filtered 5%aqueous polyethylene glycol phase into each well of a non-adherent 96 well plate that has been pre-coded with onic as described in the accompanying text protocol. Polyethylene glycol edition can be done manually with the multichannel pipette or robotically with a liquid handler using a pipette mix the cell suspension by gently pipetting up and down to Resus.

Suspend any cells that have settled over time. Add 20 microliters of the suspension to every other well from one column of a 384 well plate. Then turn on the liquid handler and home the pipetting head.

To register the coordinates, place the source plate, the destination plate and pipette tip boxes at defined positions on the workstation of the liquid handler. Using the automated liquid handler, select a mixing volume, roughly one third of the cell suspension volume and load one column of barrels from the pipetting head of the liquid handler with mixing pipette tips from the workstation. Use the liquid handler to mix the cell suspension in the 384 well source plate.

Then eject the tips into an empty waist tips box. Next, load one column of barrels from the pipetting head with 10 microliter dispensing pipette tips. Use the liquid handler to aspirate 0.3 microliters of the cell suspension from the source plate into each pipette tip.

Then dispense the cell suspension into the wells of one column of the destination plate containing 50 microliters of the 5%polyethylene glycol phase. Use a dispense height of 0.5 millimeters and dispense the drop at a flow rate of one microliter per second or slower. Repeat this process until all of the columns in the destination plate are seated with cells once complete, carefully remove the destination plate from the workstation and incubate the cells in a humidified environment at 37 degrees Celsius and 5%carbon dioxide for 24 hours prior to any drug treatment.

Visually confirms steroid formation in each well of the 96 well plate following 24 hours in culture. Use serial dilution to prepare a drug of interest such as cisplatin for testing. Dilute the stock drug solution into culture media so that each dilution is two times the final desired concentration.

Up to five different concentrations can be tested per 96. Well plate add 50 microliters of the drug solution into the polyethylene glycol phase of each. Well use two columns of eight wells per treatment group, including a control group where media alone is added.

Incubate steroids with the drug for 48 hours at 37 degrees Celsius and 5%carbon dioxide protected from light. After 48 hours, prepare fresh drug dilutions and renew the drug by adding in additional 50 microliters of media containing fresh drug at the desired concentration. Incubate the PHE for another 48 hours at 37 degrees Celsius and 5%carbon dioxide protected from light.

Next, add a cell viability reagent to each well as described in the accompanying text protocol and incubate the plate for six hours at 37 degrees Celsius and 5%carbon dioxide protected from light. When the assay is complete, place the plate in a microplate reader and read the fluorescent signal at 560 and 590 nanometers excitation and emission wavelengths respectively. High throughput screening of anti-cancer drugs requires a repeatable way to form hundreds of uniformly sized steroids.

The graph shown here emphasizes the reproducibility of this method to form spheroids. In a standard 96 well plate averaging 332 microns in diameter with a standard deviation of less than 10%The variations in steroid diameters were found to be typical of a normal distribution. When the spheroids were exposed for four days to varying concentrations of cisplatin a clinical chemotherapeutic drug, they showed a dose dependent response to the treatments.

The resultant 50%lethal dose drug concentration was found to be 4.67 Micromolar drug treatment at lethal concentrations disintegrated cancer cell sps Once mastered, this technique can be completed in approximately two hours. While attempting this procedure, it is important to remember to quickly check for aqueous two phase formation prior to proceeding to steroid printing experiments. Following this procedure, other methods like cryosectioning of steroid immunohistochemical staining of samples and other biological assays can be performed in order to answer additional questions regarding molecular targets of drugs in cancer cell steroids.

After watching this video, you should have a good understanding of how to form 3D cancer cell cultures in a convenient way and perform drug testing in a high throughput manner using standard plates and devices.