Obtaining Primary Ovarian Cancer Cells from Solid Specimens: A Method to Culture Epithelial Ovarian Cancer Cells from Ovarian Tumor Specimens

Overview

In this video, we describe a method for isolating and characterizing primary ovarian cancer cells from solid ovarian tumor tissue specimens. The epithelial cancer cells obtained using this protocol are suitable for understanding the processes that may lead to ovarian cancer initiation and development.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Tissue Processing

1. Working under sterile conditions, transfer the samples onto a Petri dish (60 mm x 60 mm) containing 10 ml of fresh, ice-cold PBS and using a sterile razor blade, further cut into the smallest pieces possible (2 mm or less) (Figures 1A and 1B).
2. Transfer the minced tissues into a 15 ml conical tube containing 10 ml of prewarmed (37 °C for 30 min) dispase II (2.4 U/ml) in DMEM and incubate at 5% CO₂ and 37 °C for 30 min. To ensure optimal digestion of the specimens, manually agitate the cell slurry every 5 min.
3. After 30 min incubation, transfer (using a 10 ml serological pipette) the cell slurry onto a cell strainer (70 μm mesh) placed on top of a 50 ml conical tube and apply a gentle pressure against the mesh using a syringe plunger. Discard any undissociated tissue (remaining on the top of the mesh) and collect the obtained cell suspension in the 50 ml sterile conical tube. Centrifuge at 320 x g for 7 min at 4 °C (Figure 2).
4. Discard the supernatant and resuspend the cell pellet in 10 ml of DMEM containing 10% FBS.
5. Incubate the cell suspension in a Petri dish at 5% CO₂ and 37 °C (Figure 3).
6. Change the medium after 24 hr from the initial plating. This allows for removal of cellular debris and the majority of the erythrocytes present in the culture.
7. Change the medium every three days for the following two weeks, after which the cultures of primary EOC are ready for downstream applications.

Representative Results

Figure 1. Processing of clinical specimens. A) Solid specimen transferred onto a Petri dish containing 10 ml of fresh, ice-cold 1x PBS. B) Clinical specimens further cut into pieces about 2 mm in size

Figure 2. Separation of EOC cells from undissociated tissues. Following enzymatic digestion, clinical specimens are transferred onto a cell strainer and a gentle pressure is applied on the digested clinical specimens by using a syringe plunger. This allows for mechanical detachment of EOC from connective tissues and recovery of EOC cells

Figure 3. Plating of resulting EOC cells suspension. After removing any undissociated tissues, the cell suspension is centrifuged and resuspended in 10 ml of DMEM containing 10% FBS and incubated in a Petri dish at 5% CO2 and 37 °C

Materials

Name	Company	Catalog Number	Comments
1x PBS, sterile	Invitrogen	14190-144
Screw lid, polypropylene specimen container, sterile	Thermo Scientific
DMEM medium, sterile	Invitrogen	11995-065
Fetal bovine serum, sterile	Thermo Scientific Hyclone	SH30396.03
100x Penicillin-streptomycin, liquid	Invitrogen	15140-122
Dispase II, sterile	Roche	04942 078 001
Small fine-tip forceps and scalpel blade, sterile	Fisher Scientific	forceps: 08-953E, blades: 08-927-5D
Small dissecting scissors, sterile	Fisher Scientific	08-945
15 ml Conical capped tubes, sterile	BD Falcon	352097
50 ml Conical capped tubes, sterile	BD Falcon	352027
10 ml Serological pipette, sterile	Corning	13-678-11E
6 ml Syringe plunger, sterile	Tyco Healthcare	8881516911
Nylon filter (70 μm ) cell strainer, sterile	BD Falcon	352350
5 cm Petri dish, sterile	Corning	430166
10 cm Petri dish, sterile	Corning	430167