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.
1. Three-dimensional Culture of Breast Cancer Cells in Basement Membrane Matrix (The Embedment Technique)
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) Cells were trypsinized. F) Cells were resuspended into a 15 ml conical tube. G-H) Cells (present in conical tube) were spun down at 100 x g for 3 min in a tissue culture centrifuge. I) Cell pellet. J) Cells (that have been spun down) were resuspended in 1 ml of FBS-supplemented RPMI medium. K) Cells were counted using a hemocytometer. L) 2.5 x 104 cells aliquoted into the microcentrifuge tube and topped off using appropriate media so as to obtain total volume of 50 μl. M) Mix cells from step 1.7 (25,000 cells in 50 μl) with the matrix-containing microcentrifuge tube from step 1.5 in a 1:1 ratio; final volume will be 100 μl. N) Gently plate 100 μl of the matrix: cell mixture from step 8 onto the solidified matrix-coated dish from step 1.2. O) Once the matrix:cell mixture is solidified, add 2 ml of FBS-supplemented RPMI media to the dish. P) Place the dish in the incubator where it will be stored for the remainder of the experiment.
Figure 2. Image acquisition and illustration of representative images. A) Images taken with a microscope. B) Differential interference contrast (DIC) imaging microscope used to acquire images and subsequently images analyzed using image analysis software. C) Sample representative DIC images of MDA-MB-231 cells (taken once a day for five days) are shown. One-way ANOVA followed by Dunnett’s multiple comparison test: *, P < 0.05. Scale bar, 100 μm.
The authors have nothing to disclose.
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) |