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 ).