Mesothelial Clearance Assay: An In Vitro Technique to Quantify the Invasion Ability of Ovarian Cancer Spheroids into Mesothelial Cell Monolayers

Abstract

Source: Davidowitz, R. A. et al. In Vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis. J. Vis. Exp. (2012)

This video describes a technique to quantitatively measure the interaction of ovarian cancer spheroids with mesothelial cells, using fluorescently labeled cells and time-lapse video microscopy. This in vitro assay models the interaction between metastasizing ovarian cancer cell spheroids and mesothelial cells lining the peritoneal cavity in vivo.

Protocol

1. Ovarian Cancer Cell Spheroid Formation

1. RFP-expressing ovarian cancer cells are cultured in 10% Base Medium (a custom cell culture medium containing a 50:50 mixture of 199 and MCDB105, 10% inactivated fetal bovine serum, and 1% pen-strep). To express RFP in unlabeled ovarian cancer cells, transfect the cells with a plasmid containing RFP and select for cells expressing RFP. Alternatively, viral vectors can be used to express fluorescent proteins transiently, or cells can be pre-incubated with a red fluorescent cell tracker dye (Invitrogen).
2. Prior to forming ovarian cancer spheroids, it is necessary to prepare low-adhesion 96-w....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
OVCA433 Ovarian Cancer Cells			Gift from Dr. Dennis Slamon
ZT Mesothelial Cells			Gift from Dr. Tan Ince
Medium 199	GIBCO by Life Technologies	19950
MCDB105	Cell Applications Inc.	117-500
FBS-heat inactivated	GIBCO, by Life Technologies	10082
Pen-Strep	GIBCO, by Life Technologies	15070
96 well plates	Corning	3799
Polyhydroxyethylmethacrylate (poly-HEMA)  192066-25G	Sigma-Aldrich		For poly-HEMA solution dissolve 6mg poly-HEMA powder in 1ml of 95% EtOH
EtOH	Pharmco-AAPER	111ACS200	Dilute to 95% in dH20
Cell culture hood	Nuaire	NU-425-300
Tissue culture incubator	Thermo Fisher Scientific, Inc.	3110
Incubator for poly-HEMA plates 305	Labline Instruments	Imperial III
Tabletop centrifuge	Heraeus Instruments	75003429/01
6 well glass-bottom dish	MatTek Corp.	P06G-1.5-20-F
Fibronectin	Sigma-Aldrich	F1141-1MG
PBS	Cellgro	21-040-CV
Microscope	Nikon Instruments		Ti-E Inverted Motorized Fluorescence time-lapse microscope with integrated Perfect Focus System
Lens	Nikon Instruments		20X-0.75 numerical apeture
Halogen transilluminator	Nikon Instruments		0.52 NA long working distance condenser
Excitation and emission filters	Chroma Technology Corp.		GFP Ex 480/40, Em 525/50 RFPmCherry Ex 575/50 Em 640/50
Transmitted and Epifluoresce light path	Sutter Instrument Co.		Smart Shutters
Linear-encoded motorized stage	Nikon Instruments
Cooled charged-coupled device camera	Hamamatsu Corp.	ORCA-AG
Microscope incubation chamber with temperature and CO2 control			Custom Made
Vibration isolation table	TMC
NIS-Elements software	Nikon Instruments		Version 3