Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

The overall goal of this procedure is to improve the scalability and reproducibility of uniform spheroid formation using a liquid overlay technique in 384 well plates. The main advantage of this technique is that it reduces excessive medium re-operation from multiple plates, and thereby improving the reproducibility of spheroid formation. We have developed and validated the method of culturing 3D spheroids for arthropod screening tests in our laboratory and our study was to evaluate what is the edge effect in the plates of the multicellular cultures.

Today I will demonstrate the procedure together with Sona, a poster from the laboratory. Begin this procedure by weighting out zero point seven five grams of low melting point agarose and add it to 100 milliliters of mccoy's 5a medium with or without phenol red and without serum. Heat the solution in a microwave and swirl it every one to two minutes to completely dissolve the agarose.

Then autoclave the solution to sterilize it. After allowing the autoclaved agarose to cool to about 70 degrees Celsius, filter it through a 500 milliliter zero point two two micrometer bottle top filter in a laminar flow box. Next, if the entire solution will not be used at once, aliquot it.

Store this ready to use agarose solution aseptically in a cold room or four degree Celsius fridge for up to four weeks. Working in a flow box, install a plastic or metal-tipped dispensing cassette in a combi reagent dispenser. Place the tubing weight into a vessel of 70 percent ethanol and then prime the cassette.

Then, move the tubing weight into a vessel of PBS and prime it again. Next, heat a stored aliquot of filtered agarose solution in the microwave to melt it. Prime the dispensing cassette with agarose solution and then start the protocol to coat 384 well tissue culture treated micro plates with 15 microliters of filtered agarose solution.

Allow the agarose in the plate to cool for 15 to 20 minutes before either seeding the cells as described in the next section of the video, or storing them at four degrees Celsius and away from direct light. Finally, to clean the dispensing cassette, place the tubing weight in 70 to 80 degree celsius sterile water and press the prime button on the dispenser. This will remove any remaining agarose in the cassette tips and the tubes.

The following cell seeding process should be performed under sterile conditions and in a laminar flow box. Begin by removing the required number of agarose coated 384 well tissue culture treated microplates from cold storage and allowing them to equilibrate to room temperature for 15 minutes. In the meantime, prepare the combi reagent dispenser for cell seeing by priming a standard tube dispensing cassette with 70 percent ethanol and sterile PBS.

Next, using the manual setting buttons on the dispenser, adjust the seeing volume to the required microliter and the dispensing speed to medium. Use recombinant cell dissociation enzyme to remove adherent human colorectal carcinoma HCT116 cells from a tissue culture flask. In a sterile beaker, make a cell suspension stock with cells at a density of 2.5 time 10 to the fourth cells per milliliter per well in 50 microliters of complete growth medium.

If more than one 384 well plate will be seeded, use a magnetic stirrer to prevent them from settling to the bottom of the beaker. Using a dispenser, add the 25 hundred cells to each well of a 384 well plate. Allow the plates to rest for 30 minutes at room temperature.

Meanwhile, take an evaporation reducing environmental microplate lid and using a five milliliter pipette, dispense four milliliters of five percent dimethylsulfoxide into the left side trough, sweeping slowly up and down. Repeat this process with the right side trough. Ensure that the liquid added to the side troughs does not merge at the center of the lid and leave a gap for gas exchange.

If too much liquid is added, it will seep into the exterior of the lid and subsequently enter the outer well of the 384 tissue culture plate. After 30 minutes has passed, examine the cells under a microscope. Resting of the plates for 30 minutes allows the cells to settle down completely in the well bottom and close to each other, which is important for the formation of single spheroids per well.

Centrifuge the seeded plates for 15 minutes at four times G.After the spin, fill the liquid reservoir of the 384 tissue culture plate with sterile water and replace the regular plate lids with the liquid filled environmental lids. Place the plates in a 37 degree celsius rotary incubator with 95 percent humidity, five percent CO2, and 20 percent oxygen and allow the cells to aggregate into multicellular tumor spheroids or MCTS's for four days. Avoid opening the incubator door for too long in the subsequent days so that the humidity level does not drop abruptly.

On day four following NCTS formation, use an automated microplate washer dispenser to add 30 microliters of prewarmed medium to each well. Place the MCTSs back in the incubator and allow the MCTSs to grow until they attain the desired size for experimentation. Every three days, to replace the medium, empirically adjust the Z height of the washer manifold and set the dispensing speed and the speed at which the wash manifold travels down into the wells at the lowest rate to minimize turbulence in the wells.

Aspirate 30 microliters of medium per well and replace it with 30 microliters of fresh, pre-warmed medium. Then return the cells to the incubator. Image the MCTSs in a high content automated imaging system using a four X air objecting with a numerical aperture of 16.

Using the imaging software, set the exposure time to 11 milliseconds and the binning to four by four. Adjust the number and spacing of the z-stacks and the pixel binning as desired for the experiment to capture an entire MCTS in each well. Process the 2D images stepwise to measure the MCTS area, major and minor axis, perimeter, and solidity.

Please see the accompanying m-code and text read me files for specific instructions. To determine the potential applicability of the routine, seven day old MCTSs were treated with different concentrations of the anti-cancer drugs, Paclitaxel, PTX, Vincristine, VCR, Oxalyplatin, OXA, Doxyrubicin, DOX, and five flurouracil, 5-FU, at three different concentrations for four days. Images of the MCTSs are shown here.

These images were analyzed to determine the area, major and minor axes, perimeter, and solidity of drug treated MCTSs compared to controls, CTLs. The major and minor axes were used to calculate the geometrical volume. There was a concentration dependent decrease in MCTS area and volume after drug treatment.

Although point zero one micrograms per milliliter of Vincrystine and zero point four micrograms per milliliter Doxyrubicin and five Fluorouracil resulted in an increase in MCTS area, the volume was significantly increased only following point zero zero one micrograms per milliliters Vincrystine. MCTS perimeter was significantly different only at the highest concentrations of Packlitaxel, Doxyrubicin, and Five Fleurouracil which completely affected the MCTS size. Treament with Packlitaxel and Vincrystine at zero point two five micrograms per milliliter and zero point zero six micrograms per milliliter and Doxyrubicin and Five Fleurouracil at 100 micrograms per milliliter, resulted in a significant decrease in the solidity, indicating a complete to partial disintegration of the MCTSs.

Taken together, these data demonstrate the utility of this method in investigating potential anti cancer drugs. Once mastered, this technique can be done in two hours if performed properly. When attempting this procedure, it is important to note that not all cell lines are suitable for a particular 3D culture method and different cell lines form spheroids that show differences in spheroid shapes, size, histology, and growth kinetics.

The stimulatory routine that we have developed allows effective evaluation of spheroid size, particularly in partially disintegrated drug treated spheroid that do not have a clearly defined boundary for the measurement of cross sectional area. After watching this video, you should have a good understanding of the fact that modification present here does not require any additional equipment or consumables and can be routinely implemented in your arthropod screening lab.