The overall goal of this procedure is to evaluate the stem cell properties of human ovarian carcinoma cells using multi and single cell based spheres assays. This is accomplished by first selecting SOX two reporter RFP lentivirus Transduced cells by Pur Mycin treatment in the second step, the RFP positive and RFP negative cells are isolated and plated by facts as single or multi-cell colonies per well. Next, the primary sphere formation is analyzed microscopically and then the primary sphere cultures are dissociated and replated for analysis of the secondary sphere formation.
Ultimately, these sphere assays are used to calculate the proportion of putative ovarian cancer stem cells that display sphere forming capacity. This method can help answer key questions in the cancer stem cell field, such as what are some potential cancer stem cell populations and what is the frequency was in the whole tumor bag. Although this method can provide insight into ovarian cancer stem cell identification, it may also be applied for isolating cancer stem cells from other solid tumor entities such as breast carcinoma.
24 hours after transducing human ovarian carcinoma cells with lentiviral particles, remove the viral supernatant and wash the cells in PBS culture. The cells in complete medium for 48 hours. Then change the medium and add 10 micrograms per milliliter of piro mycin after five days.
Treat the cultures with shield one at a one to 1000 dilution the next day. Wash the cells in PBS and then detach them with 0.05%tripsin EDTA. After three minutes, stop the reaction with complete medium.
Count the cells and centrifuge them for five minutes at 300 times G and room temperature. Resuspend the cells carefully in 0.5 to one milliliter of sterile PBS and then strain the resulting cell suspension through a 40 micrometer filter. Adjust the cells to a five times 10 to the six cells per milliliter dilution, and then add 100 microliters of spheres medium to each well of an ultra low attachment 96 well plate next for a single cell based sphere assay.
Use the single cell mode to sort one cell into each well of the plate. Then microscopically score the cells to assess the plating efficiency. Incubate the cells in fresh spheres, medium at 37 degrees Celsius, and 5%CO2 with daily basic fibroblast growth factor and epidermal growth factor supplementation.
After one week, determine the number of emerging tumor spheres by light microscopy and the presence of the fluorescent signal from the integrated reporter system by fluorescent microscopy. Count the spheres with a diameter exceeding 100 micrometers as large spheres and the spheres with a diameter of 50 to 100 micrometers as small spheres. Taking care to count real spheres and not cell clusters.
Then calculate the proportion of sphere forming RFP positive or RFP negative cells according to the formula to passage. The spheres begin by transferring the contents of each well into individual 1.5 milliliter micro centrifuge tubes. Then rinse each well three to five times with PBS to be sure to collect all of the spheres and spin down the cells for 10 minutes at 300 times G and room temperature.
After centrifugation, remove the snat and resus suspend the pellets in 200 microliters of 0.05%tripsin EDTA at 37 degrees Celsius in a soft shaker for five minutes. If no large spheres are visible after five minutes, gently tritrate the cell solution with a 100 microliter pipette tip and add 500 microliters of complete medium to inactivate the trypsin. Spin down cells resuspend the pellets carefully in spheres medium and drain the resulting cell suspension through a 40 micrometer filter as just rated.
Finally, for serial replating of the single cells, seed single cells from one primary sphere in 100 microliters of spheres, medium per well in a new ultra low attachment 96 well plate. Then use the same formula as for the primary spheres assay to assess the proportion of sphere forming cells in the secondary spheres. Assays in multi-cell based spheres assays nearly 40%of the RFP positive human ovarian carcinoma cells compared to only 20%of the RFP negative cells gave rise to an individual tumor sphere in the primary spheres assay.
Surprisingly, higher numbers of spheres are counted from lower numbers of plated cells in both MGEM and D-M-E-M-F 12 based media demonstrating that the plating modalities highly biased the results in this assay. In contrast, when cells are immobilized by the addition of 1%methylcellulose, the efficiency of the sphere formation is mostly independent of the cell density in multi-cell based spheres cultures. Approximately 35%of the cells from the RFP positive spheres lose their red fluorescent signal after seven days of culture, suggesting that these cells have undergone differentiation while over 65%of the cells retain an RFP signal suggesting a self-renewal capacity.
In contrast, cells from spheres generated from initially RFP negative cells remain RFP negative, indicating that they cannot reestablish stem cell potential under these conditions. Fluorescence microscopy performed on spheres generated from single cells confirms these data demonstrating that single spheres derived from RFP positive cells contain both RFP positive and RFP negative cells. Whereas spheres derived from RFP negative cells retain their lack of red signal Following this procedure.
Other meers like antibodies, staining or gene expression analysis via quantit PCR can be performed to further characterize the cells with severe forming capacity. While attempting this procedure, it's important to remember that in vivo transplantation assays in immunosuppressed mice need to be performed to validate the tumor stem cell properties of the sphere initiating cells.
