Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

This technique can be used to generate cell spheroids from individual and multiple cell types using a lab-on-a-CD platform, a pioneering system for the rapid generation of spheroids. The technique uses centrifugal force to deposit cells into designated microwells, allowing the sequential elation of multiple cell types for the production of diverse spheroids. For CMS culture chip fabrication, first make polycarbonate molds for the top and bottom layers of the culture chip by computer numerical control machining.

Next mix PDMS base and PDMS curing agent at a 10 to one ratio for five minutes before placing the mixture in a desiccator for one hour to remove air bubbles. Pour the degassed PDMS mixture into the CMS culture chip molds, and place the molds into the desiccator for one more hour to remove any air bubbles. At the end of the second degassing, cure the mixture in a heat chamber at 80 degrees Celsius for two hours before placing the chips in a vacuumed plasma cleaner with the surfaces to be bonded facing up.

Expose the culture chips to air-assisted plasma at a power of 18 watts for 30 seconds, and bond the two layers of the chip in the heat chamber at 80 degrees Celsius for 30 minutes to increase the adhesion strength. Then sterilize the CMS culture chip in an autoclave at 121 degrees Celsius for 15 minutes. To prepare the cells for spheroid formation, thaw a one-milliliter vial containing five to 10 times 10 to the five cells of interest in a 36.5 degrees Celsius water bath for two minutes before adding one milliliter of DMEM to the cells with gentle mixing.

Next add the cells to a 150-millimeter diameter Petri dish containing 15 milliliters of 36.5-degree-Celsius medium, and place the dish in a cell culture incubator for 24 hours. The next day, replace the supernatant with 15 milliliters of fresh DMEM, and return the cells to the incubator. To detach the cells, create the culture with four milliliters of trypsin for four minutes at 36.5 degrees Celsius and 5%carbon dioxide, stopping the reaction with fresh medium when the cells have lifted from the bottom of the dish.

To label the cells, warm an appropriate cell fluorescence dye to room temperature before adding 20 microliters of anhydrous dimethyl sulfoxide to the vial to obtain a one millimolar solution. Dilute the fluorescence to a final working concentration of one micromolar in DMEM, and add the dye to the cell suspension of interest with gentle mixing. Then place the cells in the cell culture incubator for 20 minutes.

For monoculture spheroid formation, first add 2.5 milliliters of 4%Pluronic F-127 solution to the inlet hole of the CMS culture chip while rotating the chip at 500 to 1, 000 rotations per minute. When all of the solution has been added, rotate the chip at 4, 000 rotations per minute for three minutes using the CMS system. At the end of the rotation, incubate the culture chip overnight at 36.5 degrees Celsius at 5%carbon dioxide.

The next morning, remove the Pluronic solution, and wash the culture chip with DMEM. After drying the chip for 12 hours on a clean bench, add 2.5 milliliters of the medium to the inlet port, and rotate the chip at 4, 000 rotations per minute for three minutes. At the end of the rotation, replace 100 microliters of the medium in the inlet port with 100 microliters of the prepared cell suspension while the chip rotates at 500 to 1, 000 rotations per minute.

Then rotate the chip at 3, 000 rotations per minute for three minutes to trap the cells in each microwell by centrifugal force before placing the chip in the cell culture incubator for three days, refreshing the medium as demonstrated every day. For the generation of concentric spheroid coculture, add the first population of cells in 100 microliters of medium to the inlet port of the chip while the chip is rotating at 500 to 1, 000 rotations per minute followed by three minutes of rotation at 3, 000 rotations per minute. At the end of the rotation, add 100 microliters of the second cell population while the chip is rotating at 500 to 1, 000 rotations per minute, and rotate the chip at 3, 000 rotations per minute for another three minutes.

When both volumes of cells have been injected, place the chip into the cell culture incubator at 1, 000 to 2, 000 rotations per minute for 24 hours. For the Janus spheroid formation, add 100 microliters of the first population of cells while the chip is rotating at 500 to 1, 000 rotations per minute followed by three minutes at 3, 000 rotations per minute. At the end of the rotation, place the chip in the cell culture incubator for rotation at 1, 000 to 2, 000 rotations per minute for three hours.

At the end of the incubation, add 100 microliters of the second population of cells while the chip rotates at 500 to 1, 000 rotations per minute before rotating the chip at 3, 000 rotations per minute for three minutes. Then place the cells in the cell culture incubator at 1, 000 to 2, 000 rotations per minute for 24 hours. For sandwich spheroid formation, first add 100 microliters of the first cell population while the chip is rotating at 500 to 1, 000 rotations per minute followed by three minutes of rotation at 3, 000 rotations per minute.

At the end of the rotation, place the chip on a rotator in the cell culture incubator for two hours at 1, 000 to 2, 000 rotations per minute before adding 100 microliters of the second population of cells while the chip is rotating at 500 to 1, 000 rotations per minute. Rotate the chip at 3, 000 RPM for three minutes, and place the chip back into the incubator for a three-hour incubation with rotation. Then add an additional 100 microliters of the first cell population while the chip rotates at 500 to 1, 000 rotations per minute, and rotate and culture the cells as just demonstrated for 12 hours.

Following the protocol as demonstrated, a six-centimeter diameter CMS culture chip can be successfully fabricated. The channel of the CMS culture chip comprises an inlet port, and central, slide, and microwell regions. Time-lapse images of two types of cell culture taken at 2, 000 rotations per minute for the first 24 hours of culture within individual CMS chips, as demonstrated, show the successful formation of spheroids.

Imaging of the microwells after three days of culture show that the spheroids demonstrate an excellent uniformity and sphericity. Cocultured spheroids with conentric, Janus, and sandwich structures can also be generated. In addition, cell cultures exposed to high gravity for seven days, followed by live/dead staining, demonstrate that most cells survive long-term culture within the chips.

The top and bottom layers of the CMS culture chip should be carefully aligned when they are bonded, otherwise the number of cells in each microwell may not be equal. This study can lower the entry barrier for coculture spheroid research and can be widely used in coculture studies, for example, for screening new drugs of interest.