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

Overview

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)

1. 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).
2. 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-200 Pipetman in a spiral pattern (Figure 1C). Use caution when spreading the basement membrane matrix to avoid the formation of air bubbles. Likewise, avoid spreading the matrix to close to the borders of the glass-bottom dish to prevent meniscus formation. If inexperienced handling basement membrane matrix, then precooled the dish, P-200 Pipetman and pipettes can be on ice (alternatively left overnight in a fridge at 4 °C). This step offers additional time to spread the basement membrane matrix before solidification. Place the dish(es) in a cell culture incubator (at 37 °C with 5% CO2) for at least 30 min to enable the basement membrane matrix to solidify (Figure 1D).
3. While the matrix is undergoing solidification, trypsinize a 70-80% confluent 100-mm plate of cells. Once the cells have begun lifting off of the plate, resuspend them in 10 ml of RPMI 1640 medium containing 10% (v/v) fetal bovine serum (FBS), to inactivate the trypsin (Figure 1E). Then transfer the resuspended cells into a 15 ml conical tube (Figure 1F).
4. Spin the cells (present in conical tube) at 100 x g for 3 min in a dedicated cell culture centrifuge (Figures 1G-H).
5. While the cells are being spun down, aliquot 50 μl of matrix into an 1 ml microcentrifuge tube (note: each glass-bottomed dish is allocated one microcentrifuge tube; hence, if the experiment requires 3 dishes, then it follows that 3 microcentrifuge tubes are necessary as well), and then place the tube on ice (Figure 1B).
6. Aspirate the medium from the conical tube from step 1.4, whilst leaving the pellet undisturbed (Figure 1I). Resuspend cells (that have been spun down) in 1 ml of FBS-supplemented RPMI medium (Figure 1J).
7. Once the cells are counted using a hemocytometer (Figure 1K), or a cell particle counter aliquot 2.5 x 104 cells into the microcentrifuge tube and top it off using appropriate media so as to obtain total volume of 50 μl (Figure 1L).
8. Mix the 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 (Figure 1M).
9. Gently plate 100 μl of the matrix:cell mixture from step 1.8 onto the solidified basement membrane matrix-coated dish from step 1.2 (Figure 1N). This allows for cells to be embedded in basement membrane matrix.
10. Transfer the dish(es) into the cell culture incubator (at 37 °C with 5% CO2) and allow the matrix:cell mixture to solidify for at least 30 min.
11. Once the matrix:cell mixture is solidified, add 2 ml of FBS-supplemented RPMI media to the dish (Figure 1O) and take the dish back the incubator where it will be stored for the remainder of the experiment (Figure 1P).
12. Change media every day for a period of 5 days (or as per required for an assay; the duration of the assay for MDA-MB-231 cells is 5 days in length).
13. Using a light microscope, take differential interference contrast (DIC) images of the MDA-MB-231 colonies suspended in basement membrane matrix (Figure 2A). Image 20 representative areas at 10X objective once a day for five days to determine colony morphogenesis (Figure 2C).
14. Analyze images blindly to determine cell colony stellate formation (Figure 2B). A colony is deemed to be stellate if one or more projections from the spheroid of cells are perceived. To determine the percentage of invasive stellate colonies, divide the number of stellate colonies by the total number of cell colonies per acquired image, and then average the stellate colonies percentages of the 20 images for each day.

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) 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.

Materials

Name	Company	Catalog Number	Comments
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)