Rapid generation of high-throughput 3D MTSs using an optimized
liquid overlay method
To efficiently and economically prepare size-controllable multicellular
tumor spheroids, key factors such as cell seeding density, serum
concentrations, external forces, and medium additives were
systematically optimized
(Table
S2 ). Based on the liquid overlay, cells were seeded on the
low-attachment plate coated with non-viscous polymer such that the
interaction between cells was greater than the interaction between cells
and matrix, and the cells spontaneously gathered into clusters (Costa,
de Melo-Diogo, Moreira, Carvalho, & Correia, 2018). The well plates had
different shapes, and the effect of tumor cells aggregation in the ”U
shaped” plates was better than that of the ”flat bottom” plates
(Table S2 ). In the ”flat-bottom” plates, the cells in each well
gathered into multiple cell clusters
with uneven shapes and sizes,
while in the ”U shaped” plates, the cells in each well could aggregate
into a single cell spheroid each well under the gravity and interaction
of the cells.
Due
to the oxygen and metabolite concentration gradients caused by mass
transfer limitations, MTSs with a diameter over 500 μm could establish
three main cell layers, namely the proliferating cell layer on the
surface, the resting cell layer in the middle, and the necrotic core
have hypoxic necrosis, which has been reported to as important
biological characteristics of MTSs
(Hirschhaeuser et al., 2010). The
size of MTSs was closely related to the seeding density and the culture
time. By recording and observing MTSs every day, we found that the
growth of MTSs reached the plateau on the 6th day, on which the MTSs
were chosen for the follow-up experiments. The initial seeding density
directly determined the final diameter of the MTSs. The diameters of the
MTSs on the 6th day were 182.54 μm, 367.21 μm, 512.34 μm, 883.43 μm,
1050.31 μm, with the margin of error no more than 10 %, when the
seeding densities were 500, 1000, 2000, 10000 and 20000 cells/well,
respectively (Table S2 ).
Serum contains lots of cytokines, collagen, and adhesion factors, which
have significant effects on the growth and proliferation of cells
(Brown, Bahsoun, Morris, & Akam, 2019). As shown in Table S2 ,
the low concentration of FBS was detrimental to the formation of MTSs.
Under the condition of 5% FBS, the contact between cells was impaired
and thus unable to gather into clusters. Compared with 10% serum,
increasing the FBS concentration (15%, 20% FBS) could appropriately
improve cell aggregation, but was not significant. Considering the cost,
10% FBS was chosen.
The traditional liquid overlay method would promote cells aggregation by
introducing external forces to enhance the contact between cells(Costa
et al., 2018). Therefore, we investigated the formation and growth of
MTSs under the conditions as described in Materials and
methods . Compared with the control group, the hanging drop method had
poor reproducibility with difficulty to form a single uniform cell
spheroid. Besides this, the medium was easy to volatile; EP tube
centrifugation significantly improved cell aggregation and
reproducibility, which suits lab-scale preparation of 3D MTS for
preclinical research, but it was time-consuming and difficult to
translate to large-scale production; as for rotary shaker method, the
cell clusters were gradually compacted and denser, but it was difficult
for the cells to grow and pelletize; by contrast, the whole plate
centrifugation significantly improved the roundness and repeatability of
MTSs, and it was easy to operate.
To further promote the growth of MTSs, we tested the additives in the
culture system. Basement membrane extracts such as Geltrex™ and
Matrigel™ contain abundant ECM protein, such as cytokines, laminin and
collagen, etc., could promote tumor cell growth, proliferation and
invasion, and are usually used in the construction of 3D models for
animal cells (Benton, Arnaoutova, George, Kleinman, & Koblinski, 2014;
Carey, Martin, & Reinhart-King, 2017). Therefore, we evaluated the
effect of 2.5% Geltrex™ and 2.5% Matrigel™ in the formation of MTSs.
We found that 2.5% Geltrex™ exerted no significant effect on cell
aggregation and growth, while the 2.5% Matrigel™ had extremely obvious
promoting effects on the roundness and uniformity of MTSs, and also
improved the growth of MTSs (Table S2 ).
In summary, through the screening of MTSs culture conditions, we have
established a rapid MTS formation protocol that allows to produce
uniform and controllable MTSs. In brief, the cell suspension was diluted
to 104 cells/mL, which was fully mixed with 2.5%
(v/v) Matrigel™, and then a volume of 200 μL of this cell suspension was
added to each well of ”U-shaped” low-attachment 96-well plates. The
spheroid formation was initiated by centrifuging the plate at 1000 g for
10 min. Afterwards, the plates were cultured in an incubator at 37 °C
with 5% CO2 and 95% humidity for 6 days. In this
study, the protocol was exactly followed in the formation of
multicellular tumor spheroids, unless otherwise specified. In addition
to Hela cells used in the follow-up study, other tumor cell lines (colon
cancer cells HCT116, liver cancer cells HepG2, breast cancer cells
MCF-7, lung cancer cells A549, bladder cancer cells 5637) and normal
cell line (human hepatocytes L-02) can form reproducible cell spheroids
following this protocol
(Figure. S1 ). Not only this, this protocol can be extended to
co-culturing model with stromal cells (such as fibroblasts UCF, immune
cells PBMC) to construct multi-component MTSs (Figure. S1 ).