Home › Cell Biology › Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time
Cell Biology JoVE (Open Access) Citable · DOI

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

DOI: 10.3791/51655-v
What you'll learn
  • ✓ Set up real-time invasion assays using spheroid and mesothelial co-cultures
  • ✓ Quantify ovarian cancer cell invasive capacity over prolonged time periods
  • ✓ Program and interpret RTCA instrument data for metastatic process analysis
  • ✓ Generate cancer spheroids and prepare mesothelial monolayers for invasion studies
Protocol

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Difficulty
advanced
Total time
~3-5 days (spheroid generation ~2-3 days, RTCA assay ~24-48 hours continuous monitoring)
Model organism
Ovarian cancer cell lines (spheroids); LP9 mesothelial cells
Biosafety
BSL-1

Steps

1
Label cancer cells with fluorescent markers

Optionally label ovarian cancer cells with fluorescent dyes to enable real-time visualization during invasion assays. This step facilitates tracking of individual cell behavior in co-culture.

▶ 02:05
2
Generate spheroids using methylcellulose suspension

Prepare uniform ovarian cancer spheroids by culturing cells in methylcellulose-containing medium. This creates three-dimensional aggregates mimicking in vivo tumor architecture.

▶ 03:24
3
Set up real-time cell analyzer invasion assay

Configure RTCA plates with mesothelial monolayers and prepare conditions for spheroid seeding. This apparatus continuously measures impedance changes reflecting cell invasion dynamics.

▶ 04:28
4
Program RTCA instrument for continuous monitoring

Input experimental parameters and measurement intervals into the RTCA software. Configure real-time data acquisition to track invasion over extended periods.

▶ 05:46
5
Establish LP9 monolayer and initiate assay

Seed LP9 mesothelial cells to form confluent monolayer in RTCA plates, then add cancer spheroids. Begin continuous real-time monitoring of invasion process.

▶ 07:25
6
Analyze real-time spheroid invasion kinetics

Interpret impedance data and kinetic curves showing spheroid attachment and invasion progression. Quantify invasion rates and identify temporal patterns of metastatic behavior.

▶ 09:49
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