The overall goal of this procedure is to isolate and characterize primary ovarian cancer cells from solid clinical specimens. This is accomplished by first collecting the ovarian tumor specimen and placing it in a Petri dish. The second step is to cut the sample into pieces and transfer the pieces into a conical tube with digestion reagents for incubation.
Next, the cell slurry is run through a filter where only the cells are collected For centrifugation, the final step is to discard the supernatant and resuspend the cells in media for incubation overnight. Ultimately, the epithelial ovarian cancer cells will grow in culture for weeks, which allows for downstream applications. This method can help answer key questions in the ovarian cancer field, such as what the best treatment option is for each individual patient.
The implications of this technique extend toward the treatment of ovarian cancer because it allows us to use more realistic cell cultures, which are highly susceptible to genetic manipulation and are more useful for drug screening tests. To begin, supplement 500 milliliters of delcos, modified eagles, medium, or DMEM with 10%fetal bovine serum or FBS and 1%penicillin, streptomycin or ps. Store the medium at four degrees Celsius.
Collect the sample from the OR and transport it on ice to the lab still inside the transported container. Place the sample in a biological safety hood. Transfer the sample from a 50 milliliter conical tube onto a 60 millimeter by 60 millimeter Petri dish containing 10 milliliters of fresh ice cold PBS.
Next, using a sterile razor blade further, cut the sample into two millimeter or smaller pieces. Then treat DMEM with disc paste tube in a 15 liter conical tube. Transfer the minced tissues to the DMEM dispa two mixture tube.
Then incubate the sample at 5%carbon dioxide and 37 degrees Celsius for 30 minutes. Manually agitate the cell slurry every five minutes to ensure optimal digestion. After incubation, transfer cell slurry through a 70 micrometer mesh cell strainer placed on top of a 50 milliliter conical tube.
Gently apply pressure against the mesh using a syringe plunger, discard any unassociated tissue remaining on top of the mesh, and collect the obtained cell suspension in the 50 milliliter sterile conical tube. Next, centrifuge. The conical tube at 320 times G for seven minutes at four degrees Celsius.
Discard the supernatant and resuspend the cell pellet in 10 milliliters of DMEM containing 10%FBS and 1%ps. Then transfer the cell suspension to a Petri dish and incubate the suspension at 5%carbon dioxide and 37 degrees Celsius. Change the medium 24 hours after the initial plating to remove the cellular debris and the majority of the erythrocytes present in the culture.
Finally, change the medium every three days for the following two weeks after which the cultures of primary EOC cells are ready for downstream applications. Immediately after plating. The epithelial ovarian cancer cells or EOC cells appear as single cell suspensions and in clumps with erythrocytes and cell debris abound.
EOC cell culture three days after plating shows semi adherent EOC cell clusters and less erythrocyte contamination. One week after initial plating, the EOC cell culture becomes a swirl like cluster of cells spreading on the tissue culture plastic. The typical epithelial cobble stone morphology can be seen in a confluent monolayer of EOC cells after 14 days in culture.
After watching this video, you should have a good understanding of how to isolate and characterize primary ovarian cancer cells from solid clinical specimens.
