Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Summary

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Abstract

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging. Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

Introduction

Despite its relatively low incidence, ovarian cancer is the deadliest of the gynecological diseases and the fifth leading cause of cancer deaths among women^1,2. This is mainly due to the lack of reliable tools and models that faithfully recapitulate the initiation and progression of the disease³. Most of our knowledge today about ovarian cancer has been possible through the use of immortalized ovarian surface epithelial cells (IOSEs), ovarian cancer cell lines and primary ovarian cancer cells recovered from ascitic fluid^4-7. Unfortunately, their use has several limitations including a number of genetic and phenotypic changes associated with immortalization process or in vitro passages and the heterogeneity of the population resulting from ascitic fluid preparation.

Therefore, primary ovarian cancer cells derived from identifiable and specific solid specimens of ovarian cancer represent a unique tool for studying ovarian cancer progression.

The main difficulties involved in the obtaining these malignant cells are due to an overgrowth of stromal cells or fibroblasts along with loss of viability and premature lack of proliferative capacity in the culture of these EOC cells. Several methods to create single-cell suspensions from solid tumors currently exist, through mechanical means or enzymatic dissociation, however certain techniques yield a greater amount of the preferred outcome⁸. Here, we show that enzymatic digestion with dispase II results in an effective recovery of viable, fibroblast-free EOC cells. The so-obtained EOC cultures are highly susceptible to genetic manipulation and are also useful in drug screening tests, indicating that these EOC cultures are suitable for many downstream applications.

Protocol

Ethics Statement 
Solid specimens of ovarian cancer were obtained from the University of Minnesota Tissue Procurement Facility (TPF) after Institutional Review Board Committee: Human Subject Board (IRB) approval.

1. Reagent Setup

1. Prepare Complete DMEM medium by supplementing DMEM with 10% FBS, and 100 units penicillin-streptomycin. Store the medium at 4 °C and warm to 37 °C prior to use.
2. Aliquot dispase II into separate volumes of 5-10 ml to avoid multiple freeze thaws and store at -20 °C in a nonfrost-free freezer.

2. Tissue Collection

1. Collect solid specimens of ovarian cancer (~5 g in size) at the time of surgery from areas macroscopically identified as cancer by a pathologist. Clinical specimens are de-identified and registered according to institutional protocols.
2. Place specimens in a sterile, screw lid, polypropylene specimens container filled with 30 ml of ice-cold PBS. Transport the specimens from the surgical pathology laboratory to the research laboratory for processing on ice, and within 30 min from specimen collection (Figure 1).

3. 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 2A and 2B).
2. Transfer the minced tissues into a 15 ml conic 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 3).
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 4).
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 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.

Results

Fresh clinical specimens of ovarian cancer are collected after surgery (Figure 1) and cut in small pieces (Figures 2A and 2B) constituting the cell slurry. This allows for optimal exposure of the specimens to the enzymatic treatment. Cell slurry is exposed to enzymatic digestion and incubated at 37 °C for 30 min. During the incubation time the slurry becomes increasingly cloudy (especially after each agitation) and this is a sign of tissue disaggregation. At th...

Discussion

A better understanding of ovarian cancer etiology and development is crucial to improving the outcome of women affected by this devastating disease. In this context, the use of established and "commercially available" ovarian cancer cell line has undoubtedly been tremendously useful. However, today we know that cancer cell lines are not representative of the human tumor they originated from in many aspects including the lack of heterogeneity^9,10.

Here, we report a rapid and ...

Materials

Name	Company	Catalog Number	Comments
1x PBS, sterile	Invitrogen	14190-144
Screw lid, polypropylene specimen container, sterile	Thermo Scientific	02 1090
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