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  • br Time lapse recording of

    2020-08-18


    2.6. Time lapse recording of the spheroid forming process
    Cells were stained with the fluorescence kits as mentioned. The formation of spheroids was tracked by a time-lapse recording system (Real Time Cell Culture Monitoring (CCM) System, Astec) operated in the fluorescence mode. Cells were imaged at low magnification (× 10) for 48 h. A total of 288 pictures were taken at 10 min intervals and assembled into movies using CCM software. The speed of the JSH-23 was determined by tracking the trajectories of single cells using an image recognition software (MetaMorph), with distance/time (μm/min) measurements taken every 10 min, and averaged for each cell in 8 h. At least 20 cells were tracked for each condition.
    2.7. Gene expression analysis
    Total RNA was extracted from MIA cells, PSCs, or co-cultured cells combination using TRIzol reagent (Invitrogen, USA). cDNA was syn-thesized using the Thermoscript reverse transcription polymerase re-action system (Invitrogen) priming with random hexamer. The quan-titative RT-PCR was performed by StepOnePlus Real-Time PCR instrument (Applied Biosystems, USA) using the KAPA SYBR Green qPCR Kit (Biosystem). The expression was represented by the value normalized to 18s RNA. All sequences of the primers used in the study are demonstrated in Table S1.
    2.8. In vitro cytotoxicity assays
    Gemcitabine hydrochloride (GEM) and nab-paclitaxel (trade name Abraxane®, Abr) were purchased from Sigma-Aldrich and Celgene Corporation (Summit, NJ). The Abraxane stock solution was prepared at 5 mg/ml in 0.9% sodium chloride according to the manufacture's instructions. To test cytotoxicity of the drug in tumor-like spheroids, cell viability was performed by the Cell Counting Kit-8 assay (CCK-8). Briefly, MIA cells, PSCs, and co-cultured MIA cells and PSCs (1:9) were seeded in 96-well TCPS plates or CS-HA coated plates. The cells were incubated for 48 h at 37 °C. For the GEM drug resistance assay, the mono-culture cells (from TCPS) or spheroids (from CS-HA coated plates) were subjected to different concentrations (0.001–20 μM) of GEM for 48 h. The number of cells seeded in each culture condition is summarized in Table S2. To determine the cytotoxicity of co-treatment of GEM and Abraxane of tumor-like spheroids, the PSCs spheroids or MIA:PSCs (1:9) spheroids were exposed to GEM (100 nM) or GEM (100 nM) in combination with various concentrations of Abraxane. After the treatment, cells were added the CCK-8 solution and incubated for 1 h for the TCPS groups or 3 h for the CS-HA coated groups. The absorbance was measured at 450 nm using a microplate reader (Spec-traMaxM5, Molecular Devices). The cell viability was presented as the percentage of viable cells in comparison with the untreated control.
    2.9. Zebrafish xenograft
    Zebrafish handling procedures were approved by the use of la-boratory animal committee at National Taiwan University, Taipei, Taiwan (Approval ID: NTU105-EL-00152). AB strain zebrafish (Danio rerio) were used for the tumor cell xenograft assay. Adult zebrafish were bred and paired under the standard condition, and the developmental stages of zebrafish embryos were defined as previously described [32]. MIA cells and PSCs were respectively stained with PKH26 and PKH67, and grown on TCPS or CS-HA substrates. On both substrates, the seeding ratio of MIA:PSC was approximately 1:9. After 48 h, cells on TCPS were detached by trypsinization, and cellular spheroids on CS-HA were dissociated by Accumax™ (Stem cells, Dayton, Ohio) digestion. Cells were collected and subjected to zebrafish xenograft. Dispersed cells (∼300 cells) were injected into the cell mass of zebrafish embryos at 4 hpf. The distribution of the injected cells in zebrafish embryos was observed by the fluorescence microscope at 24, 48, and 72 hpf. To 
    quantify the migration capacity of MIA cells in zebrafish embryo, the linear distance from the center of the initial cell cluster at 24 hpf to the final location of each MIA cell in a zebrafish embryo was measured and averaged by NIS-Elements software (Nikon) at 72 hpf. At least ten zebrafish embryos were analyzed for each group.
    2.10. Statistical analysis
    All assays were repeated independently for three times in each ex-periment. The statistical analyses was performed with the GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA). Experimental values are presented as mean ± SD. The statistical significance of gene expression assay was assessed by two-way ANOVA using the GraphPad software. The p values were indicated by the asterisks: *, p < 0.05; **,
    3. Results
    3.1. Morphology and characterization of pancreatic cancer cells and associated cells on CS, CS-HA, or PVA coated plates
    The pre-stained pancreatic cancer cell lines, AsPC-1 and MIA cells (red color), and PSCs (green color) were co-cultured on CS, CS-HA, or PVA coated plates to verify if they could undergo cell assembly on these polymer substrates. The morphology of tumor-like spheroids was ob-served by the inverted fluorescence microscope and the confocal laser scanning microscope. The AsPC-1 cells and PSCs co-cultured on PVA plates at 96 h appeared to form spheroids with a more pronounced 3D structure than those on CS-HA plates, the tumor cell spheroids were not surrounded by PSCs in the morphology (Fig. 1). When co-cultured with PSCs, the pancreatic cancer cells AsPC-1 or MIA were wrapped by PSCs, which was only observed on the CS-HA plates but not on the other polymers (Fig. 1). Because MIA cells gave rise to better core-shell morphology than AsPC-1, they were selected as the pancreatic cells used in later experiments. During the first 24 h, the mono-cultured MIA cells grew into clusters on CS-HA coated plates (Fig. 2A), while the mono-cultured PSCs cultured on CS-HA formed 3D spheroids in 24 h that were more tightly packed at 48 h (Fig. 2B). Co-cultured cells on CS-HA revealed very different morphology from the monolayer (TCPS) culture (Fig. S1, Supporting Information, SI). On TCPS, either MIA cells or PSCs alone were spread and attached, while co-cultured MIA cells and PSCs showed partially adherent and clustered morphology. On CS-HA coated plates, co-cultured MIA cells and PSCs in a ratio 1:9 formed 3D tumor-like spheroids (Fig. 2C). The core-shell structure was ob-served in each tumor-like spheroid of different seeding ratios on CS-HA coated plates (Fig. S2, SI). In 1:9 ratio, nearly all MIA cells were en-circled by PSCs. As the ratio of PSCs increased, MIA cells were more tightly surrounded by PSCs in the spheroids (Fig. S2, SI). The internal structure of the co-spheroids was better visualized by the confocal microscopy (Fig. 2D and Fig. S3, SI). The cross-sectional images of the co-spheroids at the middle depth demonstrated that PSCs were dis-tributed in the outer layer and MIA cells were located in the core re-gion. Further fusion of the co-spheroids was observed, particularly at the ratio of 1:9. The combination of 90% PSCs and 10% MIA cells in the 3D tumor-like spheroid model was intended to mimic the unique fea-ture of pancreatic cancer in vivo, where the stroma can occupy up to 90% of the tumor volume. Fig. S4 showed that MIA:PSC (1:9) spheroids on CS-HA remained to have high cell viability after 48 h of culture.