Optimization of Liquid Overlay Technique to Formulate Heterogenic 3D CoCultures models
Elisabete C. Costa1, Vítor M. Gaspar1, Paula Coutinho1,2, Ilídio J. Correia1*
1
CICS-UBI - Health Sciences Research Centre, Universidade da Beira Interior, 6200-
506 Covilhã, Portugal
2
UDI-IPG - Unidade de Investigação para o Desenvolvimento do Interior, Instituto
Politécnico da Guarda, 6300-654, Guarda, Portugal
*Corresponding author:
Ilídio J. Correia
CICS-UBI - Health Sciences Research Centre, Universidade da Beira Interior
Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal
E-mail: icorreia@ubi.pt
Telephone: +351 275 329 0055
Fax : +351 275 329 099
Short title: Optimized LOT to Formulate 3D MCTS
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Supporting Information
Correlation of spheroid size and shape overtime: After the establishment of the
optimal LOT parameters optical microscopy images were acquired overtime in order to
provide a correlation of between spheroids size and shape overtime. This correlation is
also very important to evaluate the reproducibility of spheroids formation under the
optimized conditions All the acquired images were then post-processed and analysed by
using ImageJ software (Rasband 1997), as illustrated in Figure S1. The MCTS major
and minor axis (Figure S1 B) were estimated to calculate the sphericity of 3D cellular
aggregates trough the calculation of spheroids asymmetry, by Equation S1 as
previously described by Cheng and co-workers, 2009 (Cheng et al. 2009).
Equation S1Spheroids asymmetry =
Major Axis
Minor Axis
Figure S1. Representative optical contrast microscopy images processing by ImageJ to
determinate spheroids shape and size overtime. A) Optical microscopy image of
HeLa:hFIB spheroid with 1:1, as cancer to hFIB ratio, with an initial cell number of
5000 after 12 days of culture. B) Exemplification of major and minor axis of MCTS. C)
Threshold image used for analysis of spheroids area (D).
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The results of spheroids shape evolution overtime are presented in Figure 7 of the main
manuscript. Spheroids with asymmetry values equal to 1,00 are considered spheroids
with perfect spherical shape. The spheroids obtained with this improved LOT were
compared with this perfect spheroid as demonstrated in Figure 7. In addition, the
spheroids surface was also determined at 2, 6 and 12 days of spheroids culture. For this
purpose, a threshold was performed in optical microscopy images of known scale
(Figure S1 C). After spheroids area selection pixel values were converted to µm2.
Results are shown in Figure S2.
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Figure S2. Size analysis of various spheroids produced by LOT. For spheroids production 24 well plates were covered 300 µL with agarose 1%
(w/v) with 200 rpm of horizontal stirring during 6 days, using various initial cell numbers (5000, 10000, 15000) and MCF-7:hFIB and
HeLa:hFIB ratios. Homotypic spheroids of of MCF-7, HeLa, hFIB and hFIB cultured in DMEM-HG (hFIB hg) were used as controls. *p<0.05,
n=4.
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REFERENCE
Cheng G, Tse J, Jain RK, Munn LL. 2009. Micro-environmental mechanical stress
controls tumor spheroid size and morphology by suppressing proliferation and
inducing apoptosis in cancer cells. PLoS one 4(2):e4632.
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