This article details a method for isolating epithelial ovarian cancer cells (EOCs) from ovarian tumor specimens. The process involves tissue digestion, cell separation, and culture to establish primary EOC cell lines.
Epithelial cells present on the outer surface of the ovary may undergo malignant transformation to cancerous cells that can grow into ovarian tumors.
To isolate these epithelial ovarian cancer cells or EOCs, begin by taking an ovarian tumor specimen and mince it into small pieces. Transfer the minced pieces into a tube containing a digestion buffer supplemented with the desired protease enzyme.
These proteases degrade the extracellular matrix proteins within the tissue, releasing EOC cells, erythrocytes, and a few dissociated fibroblasts into suspension.
Filter the cell slurry through a strainer to separate the suspended single cells from undissociated tissue pieces. Centrifuge the suspension to pelletize the EOC cells, erythrocytes, and fibroblasts. Discard the enzyme-containing supernatant.
Resuspend the cell pellet into an epithelial growth medium and transfer it into a culture dish. Incubate the culture to allow the EOC cells and fibroblasts to attach to the base of the dish while the erythrocytes float in the media.
Remove the suspended erythrocytes by refreshing the media. Over time, the media facilitates the selective growth of EOC cells over fibroblasts to form a monolayer of primary ovarian cancer cells.
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 2 millimeter or smaller pieces. Then, treat DMEM with dispase II in a 15-milliliter conical tube. Transfer the minced tissues to the DMEM dispase II 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 undissociated 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 x g for seven minutes at 4 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.
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