NARRATOR The overall goal of this method is to efficiently generate large quantities of robust cell spheroids by cell aggregation for their use in a variety of cell based assays. The main advantage of this technique is that it uses a non-proteinaceous water soluble matrix for cell aggregation and spheroid formation, which allows an easy and rapid isolation of the spheroids. This method can help garner new insights in cell biology about the effects of cell-cell and cell/matrix interactions on tissue growth in a three dimensional microenvironment.
NARRATOR Before preparing the aggregates, heat 50 milliliters of ultrapure water to 80 degrees Celsius, and add 10 grams of methylcellulose powder. Agitate the suspension until the particles are evenly dispersed. Then add cold ultrapure water to a final volume of 100 milliliters, and store the particles overnight at 4 degrees Celsius until the solution becomes clear and straw colored with no visible solids.
Pass the solution through a 0.45 micrometer filter to remove any undissolved fragments, and dilute 1:5 milligrams per milliliter of the filtered solution in the appropriate culture medium. Then filter the methylcellulose supplemented medium through a 0.22 micrometer strainer. Next, in a bio safety cabinet, wash a 70%confluent subculture with PBS, and attach the cells with three milliliters of Trypsin-EDTA association buffer in a cell culture incubator.
After a few minutes, inspect the cells under a light microscope at a 10x magnification. When the cells have lifted from the plate, neutralize the reaction with seven milliliters of serum supplemented culture medium. Transfer the cell solution to a fresh 15 milliliter tube, and then collect the liberated cells by centrifugation.
The most common cause of an unsuccessful spheroid formation, aside from contaminating particles, is the use of suboptimal concentrations of methylcellulose or serum for the aggregation and growth of your specific cell line. NARRATOR-Using a P1000 micropipette with a filter tip, triturate the pallet three to five times with one milliliter of the spheroid formation medium, pressing the pipette tip against the bottom of the tube at a slight angle, while slowly pipetting without expelling the entire tip contents to sheer the pellet. After counting, dilute the cell suspension to a one times 10 to the fourth cells per milliliter concentration, and rinse a sterile multichannel pipette reservoir with filtered ultrapure water to remove any dust and fibers.
Dispense the cells into the reservoir and use filter tips to transfer 100 microliters of the cells into each well of a 96 well U-bottom cell repellent plate, mixing the cell suspension periodically to prevent the cells from settling to the bottom of the reservoir. When all of the cells have been seated, place the plate in a tissue culture incubator and inspect the wells daily for spheroid formation. The cells should settle to the bottom of each well within six hours, and generally aggregate into a spheroid within 24 to 48 hours.
To confirm the formation of a successful spheroid, use a P10 tip under a light microscope to gently pipette medium over the spheroid. Properly formed spheroids will loosen from the plate and roll, confirming their 3-D structure. To embed the spheroids in collagen, use reverse pipetting to evenly coat the bottom of each well of an eight well tissue culture chamber slide with a minimum of 50 microliters of neutralized collagen per well.
When all of the wells have been covered, place the chamber slide in a 35 millimeter tissue culture dish filled with ultrapure water into a 10 centimeter tissue culture dish and incubate the 10 centimeter tissue culture dish at 37 degrees Celsius for about an hour. When the collagen has polymerized, use a P1000 filter tip to carefully transfer the preformed spheroids into 1.5 milliliter microcentrifuge tubes. It is critical that the collagen base layers are incubated until they are completely polymerized to avoid disturbing the collagen when adding the spheroids or the medium.
Narrator Collect the harvested spheroids in a tabletop micro centrifuge, and use a P200 pipette to remove the supernatants, wicking away any excess supernatant by tube inversion on a clean paper towel as necessary. Using a wide bore P200 pipette tip, immediately re-suspend the spheroids in 50 microliters of neutralized collagen and carefully transfer the spheroid collagen mixtures into each well of the eight well tissue culture chamber slide. Return the slide to the incubator until the collagen is polymerized.
After about an hour, slowly add at least 100 microliters of warmed culture medium containing the appropriate treatment of interest to each well for another incubation. Then use a fluorescence microscope to acquire optical imaging sections through the invading spheroids. Using this protocol, spheroids can be generated from a variety of cell lines.
Under the appropriate methylcellulose and serum treatment conditions, the individual cells settle and adhere together at the center of the well to form spheroids with a minimal adherence to the well bottom. Some cell lines are able to adhere to cell repellent plates in the absence or at low concentrations of methylcellulose, resulting in the formation of a spheroid surrounded by a cell monolayer. In general, higher concentrations of methylcellulose prevent the cells from adhering to the well.
But too high a concentration of methylcellulose reduces cell to cell adhesion, and prevents the cells from settling to the bottom of the well, resulting in the formation of loose aggregates and numerous satellite spheroids. Collagen embedded spheroids can be treated with a chemoattractant to induce the invasion of individual cells outward from the spheroid into the surrounding matrix. Fixed collagen embedded spheroids can be imaged by bright field microscopy to quantify the distance and number of invading cells, or by immunofluorescence microscopy to visualize the invasive structures formed by the migrating cells.
It's important to allow sufficient time for the cells to aggregate into structurally sound spheroids that are robust enough for their manipulation without fragmenting, and that can be easily collected for their use in subsequent assays. Our cell growth protocol can be further adapted for multiple cell biology assays. For example, seating the cells in mediums supplemented with high concentrations of methylcellulose can be used to assess cellanoicous resistance.
After watching this video, you should have a good understanding of how to generate three dimensional cell spheroids by aggregation, which will be a valuable tool for many cell biology applications.
