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  • br intensity and NIH T cell number in the

    2019-10-02


    intensity and NIH-3T3 cell number in the coculture. D: The linear correlation between EGFP fluorescence intensity and MCF-7 cell number in the coculture. Cell ratio used in the coculture was 1:1.
    Fig. 2. Fluorescence microscopy images of the coculture between DS-red-expressing NIH-3T3 and EGFP-expressing MCF-7 Liproxstatin-1 in 3D PET scaffold and 2D monolayer at 72 h, respectively. Total seeding cell number was 104 per well. Cell ratio between MCF-7 and NIH-3T3 cells was 1:1. Scale bar: 10 μm.
    2.7. Statistical analysis
    Each culture condition was studied in triplicate. JMP version 7.0 software was used to assess statistical significance with the threshold p < 0.05.
    3. Results
    3.1. Dual fluorescence monitoring of MCF-7 and NIH-3T3 in coculture
    The feasibility of dual fluorescence measurements for monitoring MCF-7 and NIH-3T3 cells in coculture was investigated. As shown in Fig. 1A, the fluorescence intensity of PET scaffold seeded with 104 EGFP-expressing MCF-7 cells was comparable with blank scaffold in Ds-red channel (560/590 nm). Similarly, there was no significant differ-ence in the EGFP fluorescence reading between PET scaffold with 104 and without Ds-red-expressing NIH-3T3 cells (Fig. 1B). These observa-tions indicated that the cross interference of EGFP and Ds-red signals was negligible and that cells in different fluorescence channels (green and red) were clearly distinguishable [39]. The proportionality be-tween cell number and fluorescence intensity was also investigated. Figs. 1C & D show that both EGFP and Ds-red culture fluorescence in-tensity increased with the total cell number with a good linearity (R2 > 0.9) in the range of 103–105 cells per well, which was consistent with the results from monocultures in our previous study [35]. Clearly, the dual fluorescence detection can be used to quantify cell growth of MCF-7 and NIH-3T3 cells in the coculture.
    Fluorescence microscopy imaging was used to investigate the mor-phology of MCF-7 and NIH-3T3 cells in 2D and 3D systems, respec-tively. The images were taken at 72 h of the cell culture. As shown in Fig. 2, NIH-3T3 cells on 2D surfaces spread extensively, while those in the 3D PET scaffold displayed more prolonged and fiber-like structure. 3D bridges between fibers could be formed in PET (yellow arrow), which was closer to the in vivo characteristics of fibroblasts. Similar observations were also reported in a study of cell coculture between human colon cancer cells and fibroblasts in Collagen I gel [40]. One the other hand, MCF-7 cells in the 3D matrices exhibited a more round shape with a relatively smaller size as compared to those in 2D system which showed a larger irregular shape. It should also be noted that multicellular spheroids of MCF-7 cells were formed in the PET scaffold with a well-organized structure (white arrow) after 72 h of cell culture. It is well known that 3D cellular aggregates can significantly enhance cell-cell and cell-ECM interactions, modify the drug distribution, and mimic tissue-specific stiffness and nutrient gradients [26,27]. The for-mation of tumor multicellular spheroid has also been reported in other 3D cell culture systems [41–44]. These results confirmed that the PET fiber can be used as a 3D scaffold to support the attachment and growth of both MCF-7 and NIH-3T3 cells with more in vivo-like characteristics, closely resembling the cellular architecture in tumor microenviron-ment.
    3.3. Optimization of cell ratio between MCF-7 and NIH-3T3 cells in coculture
    It has been reported that the cell ratios in coculture may have sig-nificant impacts on morphology, cell-to-cell interaction as well as drug distribution and efficacy [35]. In this study, the ratio of MCF-7 and NIH-3T3 cells in the coculture was optimized in terms of changes in cell growth and drug resistance. First, the potential growth interference between the two cell populations was evaluated in the 3D 40-MBR. MCF-7 cells, when cocultured with different amounts of NIH-3T3 cells, represented similar growth rate versus those in monoculture (Fig. 3A), suggesting that NIH-3T3 cells did not interfere the proliferation of MCF