The overall goal of this cell isolation protocol is to obtain pure and viable endothelial cells from patients suffering from colorectal carcinoma, both from the tumor and the corresponding normal colon. This method can help answer key questions in the tumor biology and angiogenesis field, such as to estimate and analyze the impact of the blood vessel system on colorectal tumorigenesis. The main advantage of this technique is that one can have a relatively high success rate in obtaining pure and viable human endothelial cells from patients with colorectal carcinoma.
To obtain the tissue specimen from colorectal carcinoma patients, use a fresh scalpel to obtain pieces of tissue which weigh about a gram. Then, use sterile forceps to transfer the fresh tissue pieces in 40 milliliters of ice-cold HBSS buffer with antibiotics in a 50-milliliter centrifuge tube. Then, use separate sterile forceps to transfer the carcinoma and the normal tissue sequentially from one centrifuge tube to another to wash in 40 milliliters of ice-cold HBSS buffer with antibiotics four times.
To remove all the fat, non-tumorous tissue, or potentially necrotic tissue parts, use sterile forceps and transfer the tissue pieces to a sterile cell culture Petri dish. Then, add three to five milliliters of the antibiotic-supplemented HBSS buffer to leave the tissue pieces moistened. Next, use a fresh scalpel to trim the tissue pieces.
Then, use a scalpel with a curved blade to mince the remaining tissue into small pieces. Transfer the minced tissue pieces into tissue dissociation tube filled with pre-warmed cell culture medium. Dissociate the minced tissue pieces using a tissue dissociator, along with a tumor dissociation kit for human tissue following manufacturer's instructions.
The first critical step is to correctly process and microdissect the surgery specimen in order to obtain tissue pieces with the correct size and tissue type. After dissociating the tissue pieces, centrifuge it at room temperature at 300 times gravity for one minute. Then, use a serological pipette to add 10 milliliters of pre-warmed DMEM Low medium to the MACS tube.
Next, pour the cell suspension from the tube on a cell strainer with 100-micrometer pore size set on top of a 50-milliliter centrifuge tube. Pipette an additional 10 milliliters of DMEM Low medium in the MACS tube to flush the additional cells from the tube through the strainer in the centrifuge tube. Once the entire cell suspension is filtered, wash the cell strainer with an additional 10 milliliters of DMEM Low medium.
Centrifuge the cell suspension at room temperature at 300 times gravity for seven minutes. Then, discard the supernatant once the centrifugation is over. Then, dissolve the cell pellet in five milliliters of antibiotics supplemented, pre-warmed EBM-2-MV medium in the same 50-milliliter centrifuge tube.
Remove the coating solution from the culture flask, and transfer the cell suspension directly to a T-25 culture flask maintained at 37 degrees Celsius and 5%carbon dioxide. When the confluency reaches about 80 to 90%add one milliliter of Accutase to detach the cells in the flask. Then, leave the cells with the Accutase for 10 minutes.
Next, add four milliliters of EBM-2-MV medium maintained at 37 degrees to the cells. Then, use an automated cell counting device with 10 to 25 micrometer range to count the cells. The second most critical step are all of the steps where the cells need to be split.
The estimation of the correct time point requires experience. If the split is too early, the cells will die. If the split is too late, the endothelial cells will attain senescence or fibroblast overgrowth will be an issue.
Next, centrifuge the cells at room temperature at 250 times gravity for four minutes, and expel the supernatant at the end of the run. Next, dissolve the cell pellet in pre-warmed FACS buffer. Then, add the FACS staining antibodies to the cells.
Mix and subsequently incubate at room temperature for a total of 15 minutes. After half of this incubation period, flick the tube once. Use a serological pipette to add two milliliters of FACS buffer to the labeled cell suspension.
Then, centrifuge the cell suspension at room temperature at 250 times gravity for four minutes, and decant the supernatant after the centrifugation is over. Then, add two milliliters of FACS buffer to dissolve the cell pellet, and centrifuge the cellular suspension at room temperature for four minutes at 250 times gravity. Then, resuspend the pellet in 500 microliters of antibiotic-supplemented EBM-2-MV medium.
Next, transfer the cells to the FACS sorting instrument. Identify triple-positive cells, and let them seed into the 24-well culture dish filled with fresh, pre-warmed EBM-2-MV medium. Culture the cells for the next 48 hours, and then renew the medium.
Continue changing the medium every two days till the confluency reaches 90 to 100%in five to 10 days. Then, expand the cell population according to the desired culture dish. Then, harvest once the confluency is reached, and proceed towards characterization.
First, the tumor specimen obtained is dissociated and seeded in a cell culture dish. The non-adherent cells are washed off, and the adherent cells are cultured to 80 to 90%confluency prior to FACS sorting. Next, FACS analysis is done to identify the population of the immunolabeled viable tumor and normal endothelial cells.
To do this, fluorochrome-coupled antibody specific triple labeling of the normal and tumor endothelial cells is done to be sorted through FACS. An average of 3.6 and 5.6%of the entire cell population sorted are tumor and normal endothelial cells, respectively. Next, to identify the expression of the endothelial cell markers, the normal and tumor endothelial cells obtained are subjected to immunocytochemistry.
The images here show CD31-positive normal and tumor endothelial cells. Then, the RNA is isolated from the CD31-positive population of endothelial cells and subjected to RT-qPCR to identify the other endothelial cell-specific markers. From the graph, expression of endothelial cell markers, such as CD105, VE-cadherin, and von Willebrand factor are also detected, but expression of markers from potentially contaminating cell populations are lacking, indicating purity of the isolated endothelial cell cultures.
Once mastered, this technique can be done in approximately three weeks if it is performed properly. Generally, individuals new to this method will struggle because it requires experience with primary endothelial cell cultures in order to estimate when and at which ratios the cells can be split or be further processed for the FACS sorting analysis. The implications of this technique extend toward therapy of colorectal carcinoma because the cell cultures can be used to identify mechanisms of tumorigenesis treatment response or resistance in colorectal carcinoma.
We first had the idea for this method when we read multiple reports in the literature pointing towards problems with the establishment of really pure and viable endothelial cells from human solid tumors. Visual demonstration of this method is critical, as the steps of tissue microdissection or processing for the FACS sorting analysis or further splitting of endothelial cells require experience in order to estimate the correct time point. While attempting this procedure, it is important to first establish a functional cooperative network with surgery, pathology, and tumor registry.
Moreover, you should be trained in exercise to work with primary endothelial cell cultures, as these cells are fragile. After watching this video, you should have a good understanding of how to isolate endothelial cells from human colorectal carcinoma and corresponding normal colon.
