Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This protocol has been optimized to make it easier to investigate the anticancer effect of Lactobacillus cell-free supernatant in multiple types of colon cancer cell spheroids. This technique is the first approach that investigates the anti-cancer effects of LCFS on colon cancer spheroids under in vivo mimicry conditions. Begin by placing an autoclave sterilized MRS agar plate in a 37 degrees Celsius hydrogen anaerobic chamber with 20 parts per million oxygen and inoculating the plate with freshly thawed Lactobacillus bacterial stock.

Add two to three milliliters of MRS broth, supplemented with l-cysteine to the tube and seal the tube with the butyl rubber stopper and tube cap. Next, use a loop to collect a single colony and place the colony into a 1.5 milliliter culture tube containing 500 microliters of PBS. Resuspend the colony homogenously within the saline and load the entire bacterial suspension into a one milliliter syringe.

Insert the needle into the center of the culture tube lid and deliver the colony into the medium. Place the culture in a shaking incubator at 37 degrees Celsius, 5%carbon dioxide, and 200 revolutions per minute. After two days, measure the OD620 of the culture on a spectrophotometer.

When the OD620 equals two, sediment the bacteria by centrifugation and wash the bacteria pellet with PBS. After the second centrifugation, resuspend the bacteria in four milliliters of RPMI 1640, supplemented with 10%fetal bovine serum without antibiotics and place the bacteria in the shaking incubator for four hours at 100 revolutions per minute. At the end of the incubation, pellet the bacteria by centrifugation and sterile filter the recovered supernatant through 0.22 micron strainer for minus 80 degrees Celsius storage.

To prepare colorectal cancer cell lines for spheroid formation, culture the cells of interest as monolayers in 100 millimeter Petri dishes in the cell culture incubator. When the cells reach a 70 to 80%confluency, wash the cultures two times with four milliliters of PBS per wash before detaching the cells with one milliliter of 0.25%trypsin-EDTA per dish. After two minutes, check for dissociation under a light microscope.

If the cells have detached, neutralize the enzymatic reaction with five milliliters of growth medium. Collect the cells by centrifugation in one 15 milliliter conical tube per cell culture and gently resuspend the pellets with three milliliters of growth medium per tube. Then, count the number of viable cells by trypan blue exclusion.

After counting, dilute the cells to one to two times 10 to the fifth cells per milliliter concentration in fresh medium and add methylcellulose to the cells to a final 0.6%concentration. Transfer the cells to a sterile reservoir and use a multi-channel pipette to dispense 200 microliters of cells to each well of an ultra low attachment 96-well round bottom microplate. Then, place the plate in the cell culture incubator for 24 to 36 hours before checking for the formation of spheroids by light microscopy.

For LCFS treatment, serially dilute the prepared thawed LCFS stock solution in fresh growth medium to 25, 12.5, and 6%concentrations and use a 200 microliter pipette to carefully remove as much supernatant as possible from each well of the plated colorectal cancer cell line cultures. Then, gently add 200 microliters of LCFS supplemented growth medium in triplicate to each set of cancer cells and return the plate to the incubator for an additional 24 to 48 hours. A methylcellulose concentration of 0.6%transforms the tested cancer cell line cells into compact spheroids, indicating that spheroids can be generated from several types of colorectal cancer using this protocol.

Spheroids cultured with 25%LCFS exhibit disrupted surfaces and a decreased cell viability in a dose-dependent manner. Indeed, LCFS co-cultured cells demonstrate higher levels of propidium iodide expression compared to viable control cells. Reverse transcriptase PCR analysis, Western blotting and flow cytometric annexin V/7-AAD analysis can also be performed to assess the effects of LCFS on cancer cell apoptosis.

Be sure to handle the spheroids gently when removing the growth medium as the spheroids can easily break, making it impossible to observe the effect of LCFS treatment. Following this procedure, cell viability assays, quantitative real-time PCR, Western blotting and FACS analysis can be performed to characterize the anticancer effect of LCFS in greater detail.