Our protocol is significant because it describes a generation of 3D tumor culture models from primary cancer cells, and this model represent real-world tumor biology better than cell lines. This approach is applicable to a variety of solid tumors. It is also cost-effective as it can be performed from beginning to end in a typical cell biology lab.
The 3D tumor models generated using this approach support research on the sensitivity and resistance of tumors to anti-cancer therapies or a combination of therapies. This approach generates 3D models from primary cancer cells. Therefore, it could identify which therapy is likely to be effective for a certain patient and thus help personalize his or her treatment.
Demonstrating the procedure will be Ella Itzhaki, a PhD student from my laboratory. To begin, take a small T25 flask with a single-cell adherent primary cell culture and remove the cell culture media. Wash the cells with PBS, and add one milliliter of 1x Accutase for three minutes at 37-degrees Celsius to prepare a single-cell suspension of adherent primary tumor cell cultures of 75 to 100%confluence.
Neutralize the Accutase solution by adding five milliliters of cell culture medium. Aspirate the cells with a 10-milliliter serological pipette, and deposit them in a 15-milliliter conical tube. Centrifuge the tube at 800g for five minutes at room temperature.
Remove the cell culture medium, and add eight milliliters of fresh cell culture medium on top of the cell pellet, then mix gently. To count the viable cells with a hemocytometer, take an aliquot of 50 microliters of the cell suspension and mix it with 50 microliters of trypan blue. Then count the live cells, and calculate the total number of live cells in the suspension.
Next, prepare 3D culture medium. And after calculating the number of cells needed for the assay and the total volume required, prepare cell suspension with the desired number of cells in 200 microliters of the 3D culture medium. Mix gently with the pipette to ensure homogenous distribution.
Transfer the suspension to a pipetting reservoir, and add 200 microliters of the cell suspension to each well of an ultra-low attachment 96-well plate with a multi-channel pipette. Centrifuge the plate at 300g for 10 minutes at room temperature to enforce the clustering of the cells, thereby improving the cell aggregation, and incubate the plate at 37-degrees Celsius in a 5%carbon dioxide humidified incubator. After centrifuging again at 300g for 10 minutes at room temperature, gently remove and discard 50%of the medium, and add 100 microliters of fresh 3D culture medium to replace the existing solution.
Repeat this step, and place the plate back in the 37-degrees Celsius and 5%carbon dioxide humidified incubator. Inspect the cells under a microscope every one to two days to monitor spheroid formation. Measure the diameter of the spheroids formed using the scale tool in the imaging software.
And once the spheroid diameter reaches 100 to 200 micrometers, perform the drug efficacy experiments. For spheroid collection, use a 1, 000-microliter pipette to collect the spheroids from each well, and deposit them into a 15-milliliter conical tube. Centrifuge the conical tube at 300g for five minutes at room temperature, and carefully aspirate and discard the supernatant using a pipette.
Add 0.5 milliliters of the cell culture medium, and resuspend the pellet well but gently. To perform spheroid counting, use a 96-well plate and draw a plus sign on the underside of a well to divide the well into quadrants. Add 50 microliters of the suspension to the well.
And with a 10x objective lens, count the spheroids manually under the microscope. Count the spheroids in each quadrant, and calculate the total number of spheroids in the well. Then calculate the spheroid concentration by doubling the spheroid count by counting volume, and calculate the total number of spheroids in the suspension.
In a new tube, prepare spheroid suspension at a concentration of 200 spheroids per 200 microliters of cell culture medium. For each drug treatment, prepare the stock of spheroids in a different tube. Calculate the total amount needed for each drug by the number of wells needed for repeats, and add the drug to the tube to the final concentration needed.
Transfer 200 microliters of the spheroid suspension into the wells of an ultra-low attachment 96-well plate, and incubate the plate at 37-degrees Celsius in a 5%carbon dioxide humidified incubator. After incubating the spheroids with the study drug for 24 to 72 hours, centrifuge the plate at 300g for five minutes at room temperature, and gently remove 170 microliters of the cell culture medium, leaving 30 microliters at the bottom of the well. Prepare the MTT solution, and add 70 microliters of the solution to each well to a final volume of 100 microliters per well.
The final MTT concentration in the well will be 0.05 milligrams per 100 microliters. In addition, prepare blank wells with MTT solution without cells. Incubate the plate at 37-degrees Celsius in a 5%carbon dioxide humidified incubator for three to four hours until a change in the color of the solution in the wells is observed.
When a change is observed, add 100 microliters of Stop solution to each well, and gently mix the content of the wells without creating bubbles. Read the absorbance of the plate in a parameter ELISA reader at a wavelength of 570 nanometers and a background wavelength of 630 to 690 nanometers. To calculate the cell viability, calculate the specific signal for each well.
Then calculate the average value of the blank wells, and subtract this value from each well. Calculate the average of the specific signals in the control wells that contain cells that were not treated with the study drug. Finally, calculate the viability of cells in each well relative to the wells with untreated cells.
The formation of spheroids from a primary colon cancer cell culture over time by the number of initially seeded cells is shown here. From this figure, it can be seen that the number of spheroids generated depends on the number of cells initially seeded in each well. Spheroids from the primary colon cancer cells after 10 days in culture with initial cell-seeding of 2, 000 per well are shown here.
Upon prolonged culture of the colon cancer spheroids, they started attaching to each other and formed clusters of spheroids and grape-like structures which prevented a homogenous culture and thus prohibited the use of the spheroids in the MTT assays. The effects of palbociclib, sunitinib, and their combination on spheroids from primary tumor cells, including colon and breast cancer, are shown here. An MTT assay was conducted on spheroids derived from colon and breast cancer cells.
The MTT signals were normalized to values from DMSO-treated cells. The values represent the means from four to eight replicates. The effects of the various treatments on cell growth were also evaluated microscopically at day zero and after three days of treatment of the spheroids derived from colon cancer and breast cancer cells.
The effects of trastuzumab plus vinorelbine and 5 fluorouracil plus cisplatin on spheroids derived from breast cancer over time are presented in this figure. The change in the diameter of the spheroids relative to day zero by the duration of treatment is shown here. Each treatment group included four to six wells with one spheroid in each well.
The average change is presented here. Spheroids are important tools for many studies beyond response to anti-cancer treatment. These studies addresses, for example, drug delivery and even basic biology questions such as cell-cell interactions.
It is critical to start a spheroid-generation process with a single-cell suspension. It is also critical to use ultra-low attachment plates with a medium containing a 5%basic membrane metrics.
