Revista Colombiana de Cancerología
Julio - Septiembre / 2023 Rev Colomb Cancerol. 2023;27(2):389-396
[Link]
NOTA TÉCNICA
Obtaining and characterization of spheroids using colon adenocarcinoma
SW480 cells
Obtención y caracterización de esferoides utilizando células de adenocarcinoma de
colon SW480
Susana Ceballos-Duque1 , María Elena Maldonado-Celis1 , Yuliet Montoya2
1
Escuela de Nutrición y Dietética, Universidad de Antioquia, Medellín, Colombia.
2
Centro de Bioingeniería, Universidad Pontificia Bolivariana, Medellín, Colombia.
Fecha de sometimiento: 20/02/2023 Resumen
Fecha de aceptación: 26/06/2023
Disponible en internet: 29/09/2023
El cáncer de colon ha sido estudiado convencionalmente mediante modelos in vitro
en monocapa para entender su biología y para identificar agentes terapéuticos,
pero no representan las células de un tumor que crece en tres dimensiones. Esta
Citation: nota técnica presenta un protocolo para desarrollar y caracterizar un modelo
Ceballos-Duque S, Maldonado-Celis ME, tridimensional de esferoides de diferentes tamaños formados con células de
Montoya Y. Obtaining and characterization of
adenocarcinoma de colon SW480 sembradas en micromoldes de agarosa que se
spheroids using colon adenocarcinoma SW480
cells. Rev Col Cancerol. 2023;27(3):389-96. dejaron crecer hasta por 21 días. Los esferoides se caracterizaron, mediante
[Link] citometría de flujo, con base en el ciclo celular, función mitocondrial y apoptosis.
Se encontró que esferoides de 400 µm de tamaño de siembra y tres días de
crecimiento representan un modelo que posee características similares a las de
Conflicts of interest:
Los autores declaran no tener conflictos de los tumores in vivo. Se propone un método de fácil implementación para evaluar
interés. agentes terapéuticos, previo al uso de modelos animales.
Palabras clave: esferoides celulares, neoplasias del colon, muerte celular, ciclo
Corresponding author: celular, potencial de la membrana mitocondrial
María Elena Maldonado-Celis
Escuela de Nutrición y Dietética, Universidad
de Antioquia, Medellín, Colombia. Abstract
E-mail: [Link]@[Link]
Colon cancer has conventionally been studied using in vitro monolayer models to
understand its biology and identify therapeutic agents; nevertheless, they do not
represent tumor cells growing in three dimensions. This technical note presents a
protocol to develop and characterize a three-dimensional model of spheroids of
different sizes, formed with SW480 colon adenocarcinoma cells seeded on agarose
micromolds allowed to grow for up to 21 days. The spheroids were characterized
by flow cytometry based on cell cycle, mitochondrial function, and apoptosis.
Spheroids of 400 µm seeding size and 3 days of growth were found to represent
a model possessing similar characteristics to those of tumors in vivo. An easy-to-
implement method to evaluate therapeutic agents is proposed, previous to the
use of animal models.
Keywords: Spheroids, cellular; colonic neoplasms; cell death; cell cycle; mem-
brane potential, mitochondrial.
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Introduction Spheroids were prepared from a culture of SW480
cells maintained in Dulbecco’s Modified Eagle
Colon cancer is one of the leading causes of cancer Medium (DMEM) supplemented with 25 mM glucose,
death (1). Traditional two-dimensional (2D) cell 2 mM L-glutamine, 10% fetal bovine serum, 100
culture, although advantageous (2), has limitations U/ml penicillin, 100 μg/ml streptomycin, and 1%
in mimicking real cellular environments (3), leading nonessential amino acids (Invitrogen, USA) to 90%
to the low predictive ability of pharmacological confluence at 37 °C and in a humidified atmosphere
compounds (4,5). Three-dimensional (3D) cell with 5% CO2 (14). Cells had viability greater than
culture methods, such as spheroids (6), have 90%, as determined by trypan blue in a Neubauer
been used as an in vitro tumor model for almost chamber. Microtissues® were used, following the
50 years and show significant similarities in drug manufacturer’s Casting, Equilibrating, and Seeding
response to animal models. Studies have found that the 3D Petri Dish® protocol. The process was
spheroids can induce cell death, DNA damage, and carried out under sterile conditions using a class
quiescence in response to anticancer drugs (7,8). II biosafety cabinet (Esco, Singapore). To prepare
Additionally, the effects of natural compounds, the microgels, 20 mg/mL ultrapure agarose (Sigma-
such as quercetin, can be assessed in both 2D and Aldrich) was dissolved in sterile 0.9% (w/v) NaCl
3D cultures (9). This study used the SW480 cell line (saline). The solution was shaken every 10 seconds
due to its resemblance to features observed in other after microwave heating (1,050 W, Abba, Bogotá,
colorectal malignancies, which reflect mutational Colombia) until completely clear. Then, 340 μL of
mechanisms in primary lesions (10). 3D cultures dissolved agarose was deposited in the micromold
have relevance in toxicology, and numerous models and cooled for 1 minute at room temperature (RT).
have been identified (11). This study aims to develop The gel was unmolded and transferred to a well of a
and characterize a 3D model of colon cancer using 24-well plate; 500 μL of culture medium was added
spheroids with SW480 cells, which generate their every 10 minutes to equilibrate the microgel. The
cell matrix without needing external support (12). medium was removed, and 75 μL of culture medium
The model is able to maintain distinctive cellular was deposited with different cell concentrations
phenotypes characteristic of tumors in vivo and is according to the desired spheroid seeding size (100
valuable for future research. µm: 125 cells/75 µL; 200 µm: 1,000 cells/75 µL; 400
µm: 8,000 cells/75 µL; 600 µm: 1,500,000 cells/75
µL) (figure 1). These are estimates only. Estimates
of spheroid diameter are based on the following
assumptions: i) the diameter of input cells is 20
Colon adenocarcinoma spheroids µm (4,180 µm3), ii) total spheroid volume equals
cell volume times the number of input cells, iii)
The SW480 cell line was used, as it was obtained the spheroid is a perfect sphere, and iv) cells are
from an adenocarcinoma, a primary tumor of a perfectly distributed to all recesses. In practice,
fifty-year-old Caucasian male affected by colon spheroid size and shape will vary depending on cell
adenocarcinoma, which has been characterized type.
and is considered part of an in vitro model of
cancer progression since metastatic cells have
been derived from it, allowing for a comparison Morphological characterization of SW480
with other studies of the differences acquired
spheroids
by the cells of a primary tumor when they
differentiate into metastatic cells, such as those
Spheroids were morphologically characterized by
derived from SW620 (13). The subject at hand has
inverted optical microscopy (IM-3FL model, OPTIKA
been extensively investigated, and the findings
Microscopes, Ponteranica, BG, Italy), and images
have been documented to demonstrate that the
were analyzed with the OPTIKA ProView software.
aberrant features of this particular entity are in
Images were captured with a 100x objective and
concordance with the predominant irregularities
at a scale of 200 µm at different time points; 20
manifested in other colorectal malignancies.
micrographs were taken for each size (figure 2A).
The genetic modifications observed may reflect
Images were analyzed with ImageJ 1.52v software
mutational mechanisms occurring in the primary
(Wayne Rasband, National Institutes of Health,
lesion cells (2).
USA). Statistics were calculated using GraphPad
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S. Ceballos-Duque et al.
Prism 9.0.0. One-way ordinary ANOVA and Tukey’s and 7 compared to day 1, while the percentage of
multiple comparisons tests were performed. senescent (G1), proliferative (S), and proliferative
Spheroids showed an increase in opaque areas cells in G2 significantly decreased on days 3 and
from day 3 of growth. Disintegration was observed 7 compared to day 1 of growth. Based on these
at the edges of the spheroids from day 14 and findings, subsequent experiments were performed
maintained until day 21. The 100 μm spheroids using spheroids of 400 μm in size, which were grown
showed a statistically significant decrease of one for 3 days.
area on days 3, 7, and 14 compared to day 1; on
day 21, the spheroids returned to their initial area
value (figure 2B). The 200 μm spheroids did not
show a statistically significant decrease (figure 2C). Mitochondrial function in spheroid cells
The 400 and 600 μm spheroids showed statistically
significant growth on days 3 and 7, compared to Fluorescent probes, such as DiOC6 and PI, can be
day 1, and a decrease in the area that remained used to assess changes in membrane potential
constant (plateau) on days 14 and 21 (figures 2D-E). and plasma membrane damage. Hyperpolarized
mitochondria accumulate more cation dye, whereas
depolarized mitochondria accumulate less (16).
The 400 μm spheroids were seeded as described
Cell cycle analysis in figure 1. After 1 and 3 days of growth, they
were mechanically disintegrated using a vortex
To determine the distribution of the cells composing shaker (Velp Scientifica, Usmate Velate MB, Italy)
the spheroids, DNA staining with propidium iodide at 8,000 rpm for 5 minutes; then, the sample was
(PI) was performed (15); 400 and 600 µm spheroids homogenized gently using a pipette. They were
were seeded. After 1, 3, and 7 days of growth, incubated with DiOC6 at 50 nM and 10 nM of PI at
spheroids were recovered and mechanically 1 mg/mL for 30 minutes in the dark at RT. Data for
disintegrated. Cell suspensions were washed with at least 10,000 events per sample were collected
PBS at RT. Subsequently, the cells were fixed for 1 by flow cytometry and analyzed using the FlowJo™
hour with cold 70% ethanol and washed again with software (v7.6.2) at 488 nm excitation; emission
PBS. The fixed cells were suspended in 200 μL of PBS was detected with a green filter at 530/15 nm
and 250 μg/ml RNAsa; 10 μg/mL PI was added. Cells for DiOC6 and 610/15 nm for PI. Statistics were
were incubated at RT for 30 minutes in the dark. calculated using GraphPad Prism 9.0.0. A two-way
Data for at least 10,000 events per sample were ANOVA and Tukey’s multiple comparisons test were
collected by flow cytometry and analyzed using performed. Figures 5A and 5B show a significant
the FlowJo™ software (v.7.6.2, Becton, Dickinson decrease in the percentage of viable cells in the
and Company, Ashland, Oregon, USA) at 488 nm 400 µm spheroids after 3 days of growth compared
excitation; emission was detected at 610/15 nm. to those after one day. In addition, a statistically
Statistics were calculated using GraphPad Prism significant increase in the percentage of apoptotic
9.0.0. A two-way ANOVA and a Bonferroni multiple cells without mitochondrial dysfunction is observed
comparisons test were performed. Figures 3A and over time.
3B show a significant increase in the percentage of
dead or dying cells in the 400 μm spheroids on days
3 and 7 compared to day 1, with a corresponding
increase in the percentage of senescent-phase cells Apoptotic cells in spheroids
on day 3 and a decrease on day 7.
During apoptosis, phosphatidylserine (PS)
The percentage of cells in the proliferative S-phase translocates to the outer membrane, which can be
decreased significantly on days 3 and 7 compared detected by annexin V, a protein with high affinity
to day 1. In addition, cells in the proliferative and selectivity for PS. Double staining of cells
G2 phase showed a decreasing trend on day 3, with annexin V-fluorescein isothiocyanate (FITC)
with a statistically significant reduction on day 7 and PI can differentiate between necrotic, late
compared to day 1. Figure 4 shows the distribution apoptotic, early apoptotic, and viable cells (17).
of cells in each phase of the 600 μm spheroid cell The 400 μm spheroids were obtained as described
cycle over the days of growth. Figures 4A and 4B in figure 1. After 1 and 3 days of growth, they were
indicate a statistically significant increase in the mechanically disintegrated as explained above and
percentage of dead or dying cells (SubG1) on days 3 mixed with 1 mL of annexin V-binding buffer (10
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mM HEPES; 0.14 M NaCl; 2.5 mM CaCl2; pH 7.4), Conclusions
4 μl of annexin V/FITC, and 10 μL of PI at 1 mg/
mL. Samples were incubated for 15 minutes at 4 A spheroid model was obtained from SW480 colon
°C in the dark. Data from at least 10,000 events adenocarcinoma cells of different seeding sizes
per sample were analyzed by flow cytometer (LSR and days of growth. Morphological analysis, cell
Fortessa™, BD Biosciences, USA), using the FlowJo™ cycle, mitochondrial function, and apoptosis
software (v7.6.2) at 488 nm excitation; emission showed that spheroids with a seeding size of
was detected at 610/15 nm for PI and 518 nm for 400 μm and 3 days of growth exhibit exponential
annexin V/FITC and analyzed. Figure 6A shows the growth with a decrease in viable cells, increase in
cell distribution in each quadrant after 1 and 3 days apoptotic cells, and no mitochondrial damage. This
of spheroid growth. Figure 6B demonstrates that suggests that this model resembles tumors in vivo.
400 µm spheroids grown for 3 days, compared to Furthermore, it should be noted that the process of
those grown for 1 day, had a significant increase in obtaining spheroids from SW480 cells is a strategy
apoptotic cells, including early and late phases, and for evaluating therapeutic agents before using
a significant decrease in viable cells, as observed in preclinical animal models.
prior experiments.
100μm
75μL 24h X35
spheroids
L
m
ll/
125 cell/spheroid
ce
0
00
0.
16
24h 200μm
mL 75μL X35
c ell/ spheroids
.000
467
1.000 cell/spheroid
3’7
33.
000
cel
l/m
L
75μL 24h 400μm
X35
spheroids
12
’7
20
.0
00
8.000 cell/spheroid
ce
ll/
m
L
24h
75μL 600μm
X35 spheroids
1,500.000 cell/spheroid
Figure 1. Cell seeding process for spheroid formation.
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Initial seed size (μm)
A
100 200 400 600
1
Growth time (days)
3
7
14
21
B C
100 μm spheroids 200 μm spheroids
Spheroid area (μm2)
Spheroid area (μm2)
Growing day Growing day
400 μm spheroids 600 μm spheroids
D E
**** **** ****
**** * **** ****
*
Spheroid area (μm2)
Spheroid area (μm2)
** **** **** *
Growing day Growing day
Figure 2. Morphological characterization of different spheroids (100, 200, 400, and 600 µm) at different growth times
(1, 3, 7, 14, and 21 days). A. Micrographic registration. B-E. Area of 100, 200, 400, and 600 µm spheroids. *p≤0.01;
**p≤0.001; ***p≤0.0001; ****p≤0.00001.
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A 600
B
400
**** **
200
* *** **
0
0 50K 100K 150K 200K 250K
** * * * *
Day 1
200
Day 2
Cell count
Cell count (%)
150
Day 7
100
50
0
0 50K 100K 150K 200K 250K
400
300 SubG1 G1 S G2
200
Cell cycle phase
100
0
0 50K 100K 150K 200K 250K
PI-A
PI-A=Propidium iodide-annexin; SD=Standard deviation; SubG1=Sub-Gap 1 phase; G1=Gap 1 phase; S=Synthesis phase; G2=Gap 2
phase.
Figure 3. Cell cycle analysis of 400 µm SW480 spheroids at three different times of growth (1, 3, and 7 days).
Approximately 35 spheroids and ≈10,000 events were evaluated for each condition. A. Representative histograms
of the distribution of events in each cell cycle phase. B. Bar chart with the standard deviation of the mean of three
independent experiments. *p≤0.01; **p≤0.001; ***p≤0.0001; ****p≤0.00001.
A B
Day 1
Cell count
Cell count (%)
Day 3
Day 7
SubG1 G1 S G2
Cell cycle phase
PI-A=Propidium iodide-annexin; SD=Standard deviation; SubG1=SubG1=Sub-Gap 1 phase; G1=Gap 1 phase; S=Synthesis
phase; G2=Gap 2 phase.
Figure 4. Cell cycle analysis of 600 µm SW480 spheroids at three different time of growth (1, 3, and 7 days).
Approximately 35 spheroids and 10,000 events were evaluated for each condition. A. Representative histograms of
the distribution in each cell cycle phase. B. Bar chart with the standard deviation of the mean of three independent
experiments. *p≤0.01; **p≤0.001; ***p≤0.0001.
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S. Ceballos-Duque et al.
A BB
Día 1 100
Día 1
80 Día 3
% de eventos
60
40
20
0
Q1 Q2 Q3 Q4
Día 3 Cuadrante
PI=Propidium iodide; DiOC6= 3,3′-dihexyloxacarbocyanine iodide; SD=Standard deviation; Q1=Quadrant 1; Q2=Quadrant 2; Q3=Quad-
rant 3; Q4=Quadrant 4.
Figure 5. Determination of the mitochondrial membrane potential of 400 µm SW480 spheroids on days 1 and 3 of
growth. Approximately 35 spheroids were evaluated for each condition. A. Representative dot plot of spheroids
(10,000 events). B. Bar chart with the standard deviation of the mean of three independent experiments. The table
shows the percentage of cells in the quadrants: Q1 (DiOC6−/PI+); Q2 (DiOC6+/PI+); Q3 (DiOC6+/PI−); Q4 (DiOC6−/
PI−). *p≤0.01; **p≤0.001; ***p≤0.0001.
A B
Q1 Q2
*** ** **
105 2,32% 2,47% 100
Día 1
Día 1 Día 3
104 80
% de eventos
103 60
102
Q3
40
0 Q4
0,597%
94,6%
20
P1
0 102 103 104 105
Q1 Q2
105 4,70% 21,1%
Día 3 0
Q1 Q2 Q3 Q4
104
Cuadrante
103
Día 1 Día 3
102
Q4 Q3
(%) SD(%) (%) SD(%)
0 63,3% 10,9%
Q1: necrosis 2.1 0.3 5.0 0.8
0 102 103 104 105
Q2: late apoptosis 2.8 0.6 21.3 1.1
Annexin-V/FITC Q3: early apoptosis 1.0 0.2 11.6 1.1
Q4: viables 94.4 0.3 61.0 2.8
PI=Propidium iodide; FITC=Fluorescein isothiocyanate; SD=Standard deviation; Q1=Quadrant 1; Q2=Quadrant 2; Q3=Quadrant 3;
Q4=Quadrant 4.
Figure 6. Apoptotic cell analysis of 400 µm SW480 spheroids on days 1 and 3 of growth. Approximately, 35 spheroids were
evaluated for each condition. A. Representative dot plot of spheroids (10,000 events). B. Bar chart with the standard
deviation of the mean of three independent experiments. The table shows the percentage of cells in the quadrants: Q1
(Annexin−/PI+); Q2 (Annexin+/PI+); Q3 (Annexin+/PI−); Q4 (Annexin−/PI−). *p≤0.01; **p≤0.001.
Revista Colombiana de Cancerología 395
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