3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

Abstract

Source: Cvetković, D., et al. Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model. J. Vis. Exp. (2014).

This video provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment.

Protocol

1. Three-dimensional Culture of Breast Cancer Cells in Basement Membrane Matrix (The Embedment Technique) Handling Matrigel basement membrane matrix: Thaw on ice overnight at 4 °C. Basement membrane matrix is liquid at low temperature but solidifies at room temperature. Keep basement membrane matrix on ice (Figures 1A-B). Cover the Confocal No.1 glass-bottom dish with 50 μl of basement membrane matrix by means of spreading the matrix using the tip of a P-2…

Representative Results

Figure 1. Three-dimensional culture of MDA-MB-231 breast cancer cells in basement membrane matrix (the embedment technique). A-B) A schematic of the experimental setup (performed in the fume hood). C) The well of the glass-bottomed dish coated with 50 μl of basement membrane matrix. D) Dish(es) placed in a cell culture incubator (at 37 °C with 5% CO2) to permit the matrix to solidify for at least 30 min. E)…

Materials

35-mm glass-bottomed Confocal No.1 culture dishes	MatTek Corporation	P35G-1.0-14-C	Precooled before use
Bovine serum albumin (BSA)	BioShop	ALB003.100	Used at 3% for IF
1.5 ml tubes	VWR	CA10011-700	CA10011-700
100-mm culture dish BD353003	VWR	CABD353003	Sterile, disposable
15 ml Falcon tube	VWR	CA21008-918	Sterile, disposable
1 ml filtered tips	VWR	10011-350	Sterile, disposable
200 μl filtered tips	VWR	22234-016	Sterile, disposable
20 μl filtered tips	VWR	22234-008	Sterile, disposable
Fetal bovine serum (FBS)	Sigma	F1051	Used at 10% (v/v)
RPMI 1640 Medium with Glutamine	Life Technologies	11875-119	Used for culturing of MDA-MB-231 cells
MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)	Lonza	CC-3150	Used for culturing of MCF10A cells
Olympus IX-81 microscope	Olympus		Used for 3D culture imaging (DIC images at 10X and 40X)