The Use of Ovarian Cancer Cells from Patients Undergoing Surgery to Generate Primary Cultures Capable of Undergoing Functional Analysis

Abstract

The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91%) cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model.

Doxorubicin Synergizes with 34.5ENVE to Enhance Antitumor Efficacy against Metastatic Ovarian Cancer

“…Routine histopathological examination of formalin-fixed paraffin-embedded tissue from each patient (carried out at the hospital) was followed, and the ascites from confirmed predominant high grade serous histological subtype was used for further experiments. Equal volume of patient ascites and warmed RPMI with 20% FBS was plated in T75 flasks (Corning, Tewksbury, MA) and incubated at 37°C, with 5% CO 2 and 95% humidified air (30). Fresh medium was replaced 3 to 5 days after initial plating, and every 4 to 5 days until the cells approached confluence.…”

Lessons from patient-derived xenografts for better in vitro modeling of human cancer

“…In view of this, cancer tissues, or cells, derived from PDX models that mimic the biological and molecular characteristics of the original cancer with high fidelity could be employed to provide clinically more relevant in vitro cancer modeling systems. More specifically, such PDX tissues could be used to represent primary tumor samples from patients, either through the maintenance of tissue explants in vitro [21,22] or through establishment of primary cell cultures [23][24][25]. In addition to better mimicking the heterogeneity and biological characteristics of the original malignancy, use of cancer tissue xenografts has the advantage that the original tissue can be serially propagated in vivo.…”

Advanced Ovarian Cancer Displays Functional Intratumor Heterogeneity That Correlates to Ex Vivo Drug Sensitivity

“…17 Briefly, 20 mL of ascitic fluid was added to 20 mL of warmed culture medium (RPMI 1640 medium supplemented with 10% FCS, 20 mM Lglutamine and 1% penicillin and streptomycin) in T75 flasks (Corning, NY) and incubated at 37-C, 5% CO 2 , 95% humidified air. The medium was aspirated, and 13 mL of warmed fresh medium was replaced on days 3 to 5.…”

“…18 Primary Culture From Solid Tumor Solid culture was performed using techniques previously described. 17 Briefly, 1-cm 3 solid tumor collected from intra-abdominal sites during cytoreductive surgery and transported to the laboratory in warmed culture media was dissected into~3-mm 3 pieces and incubated with collagenase/dispase (Roche, UK) solution (1 mg/mL in full medium) for 2 hours at 37-C on an orbital shaker (IKA-Vibrax-VKR) at 2g. The cell suspension was transferred to a universal container, centrifuged at 400g for 5 minutes, Phosphate Buffered Saline (PBS) washed, resuspended in full medium, and placed in a T25 flask for 30 minutes to allow fibroblast seeding.…”