All novel cell lines were established from either tumor tissue excised during surgery or from a cell pellet derived from ascites. Spontaneously immortal cell lines were generated using the protocols and techniques previously described. 22 In brief, the tumor tissue was diced into small pieces (~1‐2 mm3) and smeared across a 10 cm tissue culture dish or 75 cm2 flask and placed under 5 to 8 mL of DMEM media in sterile conditions. Ascites fluid was centrifuged at 4°C at 1000g for 5 minutes to produce a cell pellet which was mixed with a small volume of media (5‐8 mL) and placed in a tissue culture flask under sterile conditions. All cells were initially cultivated in DMEM medium containing 25 mM d‐glucose with 2 mM l‐glutamine (ThermoFisher Scientific Inc, Massachusetts) and supplemented with 10% fetal calf serum (Sigma‐Aldrich, Missouri) and x1 Antibiotic‐Antimycotic solution (ThermoFisher Scientific Inc). Cell lines were propagated for over 20 passages to demonstrate immortalization with a passage being performed every 2 to 5 days. Once established, the Antibiotic‐Antimycotic solution was removed from the media to demonstrate that no infection was present within the cell line.

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