This new method of spheroid cultures can be used to study the tumorigenicity to analyze the presence of different populations of cancer stem cells and also can be nicely applied to for through high put screen for new drugs. This is a robust and repetitive cultured system for generating large number of homogeneously sized spheroids. Moreover, these spheroids can be used to study cancerous stem cells.
This method is set up to study chronic cancer biology but it can be easily set up to study other cancers by changing the 3D culture conditions. Be sure to watch for bubbles in the medium, to not move the plate too much during the spheroids formation and to be careful when harvesting the spheroids into the strainer. Begin by pre treating the wells of a 24 well plate with 500 micro liters of Anti-Adherence Rinsing Solution per well.
After a few seconds centrifuge the plate in swinging-bucket rotor and rinse each well with 2 milliliters of warm Basal medium. Observe the plate under the microscope to ensure that the bubbles have been completely removed from the micro wells. If no bubbles are observed, rinse the wells again before adding one milliliter of warm DMEM basement membrane matrix medium to each well.
To induce spheroid formation, see the desired concentration of Caco-2 cells in a 2D mono layer in a 10 centimeter dish in DMEM supplemented with 10%FBS and 1%antibiotics. For the culture at 37 degrees Celsius and 5%carbon dioxide in a humidified atmosphere. When an 80%confluency is reached, wash the cells with five milliliters of PBS before treating the culture with two milliliters of Trypsin-EDTA for two to five minutes at 37 degrees Celsius and 5%carbon dioxide.
When the cells have detached neutralize the Trypsin with four milliliters of complete DMEM. After counting, collect the cells by centrifugation and resuspend the palette in the appropriate volume of DMEM basement membrane matrix medium to achieve the desired concentration of cells in one milliliter of medium per well. Add one milliliter of cells to each well of the prepaid 24 well plate, followed by the addition of one milliliter of DMEM basement membrane matrix medium to each well.
After seeding, immediately centrifuge the plate and use a light microscope to verify that the cells have been evenly distributed across the micro wells. Then place the plate in the cell culture incubator for 48 hours without disturbing the cells. To harvest the spheroids, at the end of the incubation, use a serological pipette and half of the supernatant in each well to gently dislodge the spheroids from their micro wells.
When all of the spheroids have been detached use the pipette to carefully transfer the 3D cultures from each well unto a 37 micron reversible strainer on top of a 15 milliliter conical tube. Wash each well three times with one milliliter of prewarmed Basal medium per wash to collect any remaining spheroids and add these additional spheroids to the strainer. After the last wash check the plate under the microscope to confirm that all of the spheroids have been removed from the micro wells.
Then invert the strainer over a new 15 milliliter tube and wash the bottom of the strainer with fresh DMEM basement membrane matrix medium to collect the spheroids into the tube. Spheroids are rising from 500 cells demonstrate the most homogeneous increase in size over time. With 500 and 600 cells per spheroid determine to be the optimal number of cells under which good growth and low variability can be achieved.
The addition of basement membrane matrix enhances spheroid growth and homogeneity. In long term culture, the cells appear as dense multi layers at day three. While flattened cells arranged in mono layers are clearly visible at day 10.
Interestingly, a lumen appears within the spheroids from day five onward. In addition, a clear increase in proliferating cell nuclear antigen positive cells is observed at days five and seven that declines by day 10. Surprisingly, activated Caspace 3 is observed in very few cells at days three and five only.
While high levels of Beta-catenin expression are observed at every time point. The potential of the spheroids to differentiate into Anterosites is evidenced by their Alkaline Phosphatase and solute carrier family 2A5 mRNA expression. In addition, this spheroids express typical cancer stem cell markers and respond to combination chemotherapy treatments routinely administered to colorectal cancer patients.
So spheroids are composed of different cell populations. They then can be nicely used to analyze cell specifical responses to stimuli such as refractors and eventually for drug responses.
