The Next Step in the Evolution of 3D Culture:
Utilizing Extracellular Matrix to Enhance
Multicellular Tumor Spheroid Models for
Proliferation and Invasion
Overview of methodology, tools and
reagents for evaluating cell proliferation
and invasion using multicellular tumor
spheroids.
TOOLS AND REAGENTS
3-D SPHEROID
PROLIFERATION/VIABILITY
Sample Preparation for Spheroid Formation
Calculate volumes for 10X Spheroid Formation ECM, Complete Growth Medium, and
Cells (1 x 10^6 cells/ml):
Reagent
10X Spheroid Formation ECM (4 °C)
Cells (at 1 x 10^6 cells/ml)
Complete Growth Medium (4 °C)
Total
1 Well
5 µl
X µl
45-X µl
50 µl
3,000 Cells/Well
5 µl
3 µl
42 µl
50 µl
Sample Preparation for Spheroid Formation
10X
Spheroid
Formation
ECM
Complete Growth
Medium
Spheroid Formation ECM
• Thaw 10X Spheroid
Formation ECM on ice at
4 °C overnight.
• Divide into working
aliquots; store at -80 °C
for optimal stability, avoid
freeze-thaws.
• Keep on ice throughout
procedure.
• Dilute with cold (4 °C)
complete growth
medium, and pipet up
and down to mix.
Sample Preparation for Spheroid Formation
Harvest
Centrifuge
Resuspend
Cells at
1 x 10^6 cells/ml
Cells at 80% Confluence
Spheroid Formation ECM
Cells in Spheroid Formation ECM
• Culture cells to 80%
confluence.
• Harvest cells, centrifuge,
and aspirate medium.
• Resuspend at 1 x 10^6
cells/ml in complete
growth medium.
• Add the calculated
amount of cells to the
tube containing Spheroid
Formation ECM.
Sample Preparation for Spheroid Formation
Cells in Spheroid Formation ECM
96 Well Spheroid Formation Plate
Cells and Spheroid Formation ECM in 96 Well Spheroid Formation Plate
• Add 50 µl of the cells in
Spheroid ECM to the 96
Well Spheroid
Formation Plate.
• Centrifuge at 200 x g for
3 minutes in a swinging
bucket rotor.
• Incubate plate with cells
at 37 °C, 5% CO2 for 72
hours.
Sample Preparation for Spheroid Formation
Spheroid formed within 72 hours
Add 50 µl of 2X treatment.
Incubate for desired treatment period.
• Cells assemble into one
spheroid/well within 72
hours.
• Prepare solutions
containing 2X treatment
compounds in complete
growth medium.
• Add 2X treatment (50 µl)
to each well.
• Incubate at 37 °C, 5% CO2
for desired treatment
period.
Image Analysis Evaluation of Spheroid Growth
Image Analysis
3-D Culture Spheroid Proliferation/Viability
MDA-MB-231 3-D Spheroid Growth
A
B
Area (µm2)
1000000
800000
600000
400000
MDA-MB-231 Breast cancer cells in spheroid culture for 3
days form A) loose colonies in media and B) tight colonies in
media + ECM.
200000
0
0
10
20
30
Days
2
3
6
7
8
10
13
14
21
22
24
27
29
31
35
40
• 3-D spheroid cultures are large
cell aggregates which exhibit
physiological characteristics of
small tumors .
• Incorporation of ECM proteins
is necessary to promote in vivo
cell behavior.
• This assay provides an in vitro
model for evaluating tumor
growth in response to
pharmacological agents.
300000
120%
250000
100%
200000
Area (% untreated)
Area (µm2)
3-D Culture Spheroid Proliferation/Viability
Image Analysis
y = 23918x + 163064
R² = 0.9941
150000
100000
50000
y = -0.143ln(x) + 0.7463
R² = 0.9992
80%
60%
40%
20%
0
0%
0
1
2
3
4
5
0.1
1
Day
0
1
2
10
100
Bleomycin (µM)
3
4
Spheroid growth of MDA-MB-231 breast cancer spheroids. Cells
were seeded at the corresponding concentrations in the presence of
spheroid formation ECM and incubated for 72 hours at 37 °C, 5%
CO2 to induce spheroid formation. At that time, 50 ul of complete
medium was added to each well, and spheroids were incubated at
37 °C, 5% CO2. Spheroids were photographed every 24 hours, and
images were analyzed using ImageJ software.
0.16
0.8
4
24
Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells
were seeded at 3,000 cells/well in the presence of spheroid
formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to
induce spheroid formation. Spheroids were then treated with the
corresponding doses of Bleomycin and incubated at 37 °C, 5% CO2
for 96 hours. Spheroids were photographed after 96 hours, and
images were analyzed using ImageJ software
3-D Culture Spheroid Proliferation/Viability
Analysis
Fluorometric
or
Colorimetric
Quantitation
Fluorometric Quantitation with Resazurin
3-D Culture Spheroid Proliferation/Viability
Fluorometric Quantitation
16000
120%
y = 2935.7ln(x) - 10541
R² = 0.9973
14000
100%
12000
80%
Viability
RFU
10000
8000
6000
60%
y = -0.086ln(x) + 0.8038
R² = 0.9877
40%
4000
20%
2000
0
0%
10
100
1000
10000
Spheroid Seeding Density (Cells/Well)
Spheroid cell number corresponds to fluorescence output for MDAMB-231 breast cancer spheroids. Cells were seeded at the
corresponding concentrations in the presence of spheroid formation
ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid
formation. At that time, 50 ul of complete medium was added to
each well, and spheroids were incubated at 37 °C, 5% CO2 for 96
hours and analyzed using Resazurin.
0.1
1
10
100
Bleomycin (uM)
Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin.
Cells were seeded at 3,000 cells/well in the presence of spheroid
formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to
induce spheroid formation. Spheroids were then treated with the
corresponding doses of Bleomycin, incubated at 37 °C, 5% CO2 for
96 hours, and analyzed using Resazurin.
Colorimetric Quantitation with MTT
3-D Culture Spheroid Proliferation/Viability
Colorimetric Quantitation
90%
0.400
y=
0.350
-1E-09x2
+ 4E-05x
R² = 0.9961
80%
70%
0.250
0.200
0.150
50%
40%
30%
0.100
20%
0.050
10%
0.000
0%
-0.050
y = -0.118ln(x) + 0.7563
R² = 0.9979
60%
Viability
Absorbance (570 nm)
0.300
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
Spheroid Seeding Density (Cells/Well)
Spheroid cell number corresponds to absorbance for MDA-MB-231
breast cancer spheroids. Cells were seeded at the corresponding
concentrations in the presence of spheroid formation ECM and
incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid
formation. At that time, 50 ul of complete medium was added to
each well, and spheroids were incubated at 37 °C, 5% CO2 for 96
hours and analyzed using MTT.
-10% 0.1
1
10
Bleomycin (µM)
100
1000
Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin.
Cells were seeded at 3,000 cells/well in the presence of spheroid
formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to
induce spheroid formation. Spheroids were then treated with the
corresponding doses of Bleomycin, incubated at 37 °C, 5% CO2 for
96 hours, and analyzed using MTT.
3-D SPHEROID INVASION
Sample Preparation for Spheroid Invasion
Spheroid in Spheroid Formation Plate
Invasion
Matrix
Thaw on ice at 4 °C overnight.
Chill on ice at 4 °C for 5 minutes.
Add Invasion Matrix and centrifuge 300 x g for 5 min at 4 °C.
• Cells assemble into one
spheroid/well within 72 hours.
• Chill 96 Well Spheroid
Formation Plate on ice at 4 °C
for 5 minutes.
• Working on ice, add 50 µl of
Invasion Matrix to each well.
• Centrifuge plate at 300 x g for
5 minutes at 4 °C (pre-chilled)
in a swinging bucket rotor.
Sample Preparation for Spheroid Invasion
Place Spheroid Formation Plate at 37 °C, 5% CO2.
Add 100 µl of 2X treatment (37 °C).
Incubate for desired treatment period.
• Transfer the Spheroid
Formation Plate to 37 °C,
5% CO2 for one hour to
polymerize Invasion
Matrix.
• Add 100 µl of complete
medium with 2X invasion
modulating agents (37 °C)
to each well.
• Incubate at 37 °C, 5% CO2
for desired invasion
period.
• Analyze plate.
3-D Culture Spheroid Invasion
DESCRIPTION
TIME
Resuspend cells in spheroid formation ECM
and add to round bottom wells.
Cells assemble into compact spheroids.
3 days
Add Invasion Matrix and medium containing
chemoattractants and/or invasion modulating
compounds.
Cell invade into the surrounding matrix in
Response to chemoattractants
1-2 hours
1-2 hours
2-4 days
Total Time: 5-7 days
3-D Culture Spheroid Invasion Analysis
Bright field
RFP
PKH67
PKH26
3-D Culture Spheroid Invasion Analysis
Morphology of 3D cell invasion over a four day period. Cells were seeded at 3,000 cells/well in the presence of Spheroid
Formation ECM, and incubated at 37 °C, 5% CO2 for 72 hours to form spheroids. Spheroids were then embedded in 50 µl Invasion
Matrix, and incubated at 37 °C, 5% CO2 for one hour for polymerization. Then 100 µl of DMEM, 10% FBS was added to
each well as a chemoattractant. Non-invasive cells (MCF-7) remain as cell aggregates and do not invade into the surrounding
invasion matrix; whereas, invasive cells (MDA-MB-231) invade into the surrounding invasion matrix as spindle-like protrusions.
3-D Culture Spheroid Invasion Analysis
Quantitative analysis of surface area for non-invasive (MCF-7) and invasive (MDA-MB-231) cell
lines over a four day period.
Different cell lines exhibit different invasive
potentials and morphologies
Days Post Imbedding
Cell Line
0
1
2
3
4
PC-3
MDA-MB-231
HT-1080
U-87-MG
Invasion of cancer cells out of MCTS and into the surrounding invasion matrix in a time-dependent manner. Cancer cells invade into an
invasion matrix composed of 10 mg/ml BME and 1 mg/ml collagen I in response to cell culture medium containing 10% FBS; wells were
photographed every 24 hours over a 96 hour period.
Area (µm2)
3-D Culture Spheroid Invasion Analysis
900000
800000
700000
600000
500000
400000
300000
200000
100000
0
MDA-MB-231 3-D Invasion Day 4
3-D culture spheroid
invasion may be used
to evaluate
modulators of
metastasis and EMT.
0
0.5
0
0.5
10
20
1
5
Sulforaphane (µM)
Sulforaphane (uM)
10
20
1
No
Matrix
5
No Matrix
MDA-MB-231, human breast cancer cells, were induced to form spheroids (72 hours) and embedded in Invasion
Matrix. Serial dilutions of Sulforaphane were added to the DMEM, 10% FBS invasion modulating solution as
indicated, and spheroid invasion was conducted over a 96 hour period. Sulforaphane inhibited MDA-MB-231
spheroid Invasion in a dose-dependent manner.
Summary
• 3-D cultures provide suitable environments for cells to selfassemble into organotypic structures mimicking the morphology
and function their tissue of origin.
• Spheroids provide ideal tumor models based on morphology, size,
physiological gradients, and the formation of heterogeneous cell
populations.
• Spheroids exhibit an extracellular matrix which is instrumental in
self-assembly of cancer cells.
• The addition of ECM proteins promotes spheroid formation and
growth.
• ECM hydrogels provide physiological scaffolds for cancer cells to
invade out of tumor spheroids, mimicking early events in
metastasis.
Tools for 3-D Culture Spheroid
Proliferation/Viability and Invasion
Description
Cultrex® 96 Well 3-D Spheroid BME Cell Invasion Assay
Cultrex® 96 Well 3-D Spheroid Fluorometric Proliferation/Viability Assay
Cultrex® 96 Well 3-D Spheroid Colorimetric Proliferation/Viability Assay
Size
96 samples
96 samples
96 samples
Catalog #
3500-096-K
3510-096-K
3511-096-K
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