Isolation and Culture Expansion of Tumor-specific Endothelial Cells

The overall goal of this procedure is to isolate tumor endothelial cells using magnetic columns and cloning rings. This is accomplished by first digesting tumor tissue dissected from mice to generate a single cell suspension. The second step is to enrich the endothelial cell population using a magnetic bead column.

Next, the endothelial cell colonies are grown and selected using a cloning ring method. The final step is to expand and characterize the selected endothelial cell colonies. Ultimately, immunofluorescence microscopy using specific endothelial cell markers is used to show the purity of the clonal populations.

The main advantage of this technique over existing methods like fact sorting, is that it is gentler on the cells, which allows for greater recovery of viable endothelial cells and subsequent colony expansion. This method can help answer key questions in the tumor angiogenesis field, such as how vascular endothelial cell dysfunction in tumors contributes to tumor progression. Generally, individuals new to this method will struggle because endothelial cells are frequently contaminated with other cell types from the tumor microenvironment, and thus one must be diligent in quickly removing these contaminating cells from the cultures Following euthanasia.

Bring the mouse to a clean bench and disinfect the ventral side by spraying it with an ample amount of 75%volume to volume. Ethanol next, pin the limbs of the mouse on the dissecting board using proper aseptic technique. Make a midline ventral incision without opening the peritoneum while keeping the peritoneum intact.

Dissect laterally between the skin and peritoneum towards the MA glands where the tumors are located. Excise only tumor tissue from the memory glands by carefully trimming non-tumor tissues, including skin and muscles. Place the dissected tumors in a conical tube containing 30 milliliters of low glucose D echos, modified eagles medium, or LG DMEM To begin tumor dissociation.

Transfer the tissue in sterile L-G-D-M-E-M to a tissue culture dish and wash one to two times with L-G-D-M-E-M. After the washes, add two milliliters of L-G-D-M-E-M and mince the tumors with a pair of sterile scissors. Add five milliliters of collagenase, one milliliter of disc pace, 75 microliters of deoxy ribonuclease, and two milliliters of L-G-D-M-E-M to the minced tumor.

Transfer the collagenase tissue mix from the Petri dish to a tissue dissociation tube. Using a preset dissociation program, perform two 62nd rounds of dissociation. Incubate the tube with light shaking for 75 minutes at 37 degree Celsius.

Following incubation, filter the digested tissue through a 100 micron cell strainer positioned over a 50 milliliter conical tube. Rinse the filter with five milliliters of fax buffer to collect any remaining cells and centrifuge the tube at 280 times G for five minutes. Carefully remove the supernatant without disturbing the cell pellet.

Next, dilute one milliliter of 10 x stock. Red blood cell lysis buffer in nine milliliters of sterile water and resuspend the pellet to lyse any contaminating red blood cells immediately centrifuge the cells at 280 times G for five minutes. Remove the supernatant and resuspend the cells in 10 milliliters of fax buffer.

To begin the immuno magnetic separation count live cells using a hemo cytometer and dilute to 10 to the seven cells per 100 microliters. In fax buffer, add 10 microliters of FCR blocking solution per 100 microliters and incubate on ice for 10 minutes After blocking, add rat anti mouse PE conjugated CD 31 antibody to the cells and incubate them on ice for 10 to 15 minutes. With occasional agitation, add 10 milliliters of fax buffer to the tube and centrifuge at 280 times G for five minutes.

Carefully remove the snat and wash the cell pellet with five milliliters of fax buffer centrifuge again at 280 times G for five minutes to pellet the cells and then aspirate the supernatant. Now add fax buffer and anti PE microbeads to the cells and incubate them on ice for 10 to 15 minutes. With occasional agitation, add 10 milliliters of fax, buffer and centrifuge the samples at 280 times G for five minutes.

Wash the cells with five milliliters of fax buffer centrifuge and remove the supernatant After bringing the volume to 300 microliters. With fax buffer centrifuge the cells through a 35 micron cell strainer capped tube at 280 times G for five minutes. Aspirate the supernatant for magnetic separation.

Set up a magnetic multis stand and magnetic columns. In the cell culture hood, attach a column to the magnetic separator and then equate the column with two milliliters of fax buffer. Next, re suspend the cell palate in half a milliliter of fax buffer and pass the cell suspension through the equilibrated magnetic column.

Wash the column three times with two milliliters of fax buffer while collecting the flow through in a 15 milliliter conical tube. Remove the column from the magnetic separator and elute with two milliliters of fax buffer using a plunger to ensure all of the cells are removed from the column. Collect the eluate in a new 15 milliliter conical tube.

Centrifuge the eluate fraction at 280 times G for five minutes and remove the supernatant resus. Suspend the cells in 10 milliliters of endothelial cell media equally. Divide the cell suspension into at least four 10 centimeter gelatin coated cell culture dishes and incubate at 37 degrees Celsius and 5%carbon dioxide for an expected co fluency of approximately 1%The next day.

Endothelial cell colonies should start to form after seven to 10 days and grow to three to five millimeters in diameter as colonies appear after seven to 10 days, remove any nonspecific surrounding cells with a sterile 200 microliter pipette tip. Once the colonies have grown to the correct size, view the dishes using a phase contrast microscope at four x or 10 x magnification and outline individual colonies on the bottom of the culture dish. Using a fine tip marker, wash the plate with 10 milliliters of PBS and aspirate leaving a small amount of PBS to keep the cells moist.

Using, using a pair of dissecting forceps, pick up a cloning ring and pipette approximately half a microliter of tissue adhesive to the bottom. Spread the tissue adhesive around the bottom of the cloning ring evenly with a 10 microliter pipette tip. Place the cloning ring over the endothelial cell colony using the marks as a guide.

Gently press down on the cloning ring to glue the ring onto the plate. Next, pipette 25 microliters of enzymatic cell detachment solution into the cloning ring and incubate for approximately one minute or until the cells are loosely attached. Pipette the cells from each cloning ring into one well of a six.

Well plate. Wash the ring with 50 microliters of endothelial cell media to collect the remaining cells and transfer them to the same well in the six well plate grow isolated colonies in the six well plate until they reach 80 to 100%co fluency before transferring the cells to a new dish. Immuno magnetic endothelial cell isolation alone often results in culture contamination with non endothelial cells.

Here endothelial cells specifically stained with dye, I-A-C-L-D-L can be seen commingling with unstained non endothelial cells. After using clonal rings for colony, even cell populations that have been expanded show uniform DI IAC LDL staining indicating that they are pure endothelial cell populations comparing three separately isolated colonies. Distinct uniform CD 31 positive populations are apparent.

CD 31 is an endothelial cell marker while IgG serves as an isotype negative control for non-specific antibody binding. These cell populations also exhibited increased expression of endothelial cell marker genes between 200 to 7, 000 times higher than mouse embryonic fibroblast controls. Finally isolated normal or tumor endothelial cells retain endothelial cell function as evidenced by spontaneous vessel leg structure formation in culture.

After watching this video, you should have a good understanding of how to perform a high fidelity isolation of tumor specific endothelial cells using immuno magnetic enrichment, coupled with colony selection and in vitro expansion.