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  • br Materials and methods br Preparation of the


    2. Materials and methods
    2.1. Preparation of the CS, CS-HA, and polyvinyl alcohol (PVA) coated culture plates
    The chitosan (CS) powder (510 kDa) with deacetylation degree 77% was purchased from Sigma-Aldrich (USA). The hyaluronan (HA) was a sodium salt (1800 kDa) acquired from SciVision Biotech (Kaohsiung, Taiwan). The chitosan-hyaluronan (CS-HA) coated plates were pre-pared following the procedure described in literature [29]. First, the solution was coated onto 24-well tissue culture polystyrene (TCPS) plates (0.3 ml per the CS solution was prepared with 1 wt% chitosan dissolved in 1% acetic JSH23 per well). The CS surface layer formed after the solvent (acetic acid) was air-dried in a laminar flow cabinet for overnight. The residual acetic acid was removed by soaking in sodium hydroxide (0.5 N) for 30 min, and washing with phosphate buffered saline (PBS). HA solution (3 mg/ml) was then added onto CS-coated wells (0.3 ml per well) and evaporated in a laminar flow cabinet for overnight, before further crosslinking with 0.9 ml ethyl (dimethylami-nopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution for 48 h. After crosslinking, the CS-HA coated plates were washed thoroughly with PBS to remove unbound HA and stored in 4 °C before 
    use. PVA solution (0.5% in distilled water, 0.3 ml) was dropped into each well of 24-well TCPS plates and then air dried for overnight to obtain the PVA coated plates.
    Pancreatic cancer cell lines, MIA PaCa-2 (MIA) and AsPC-1 were obtained from the American Type Culture Collection (ATCC). Human PSCs (HPaSteC, #3830, ScienCell™ Research Laboratories, Carisbad, CA) were obtained from Genomic Research Center, Academia Sinica (Taipei, Taiwan). MIA cells were maintained in Dulbecco's modified Eagle's medium-high glucose (Gibco) supplemental with 10% heat-in-activated fetal bovine serum (Gibco) and 10 mg/l Antibiotic-Antimycotic (Invitrogen). PSCs were cultured in SteCM stellate cell medium (ScienCell™ Research Laboratories). The medium used for co-culture of MIA cells and PSCs was a mixture 1:1 ratio of respective medium. All the cells were incubated in the humidified atmosphere at 37 °C with 5% CO2.
    2.3. Cell seeding and characterization of tumor spheroids
    1.2 × 105 cells were seeded on the 24-well CS, CS-HA, or PVA coated plates to examine the formation of tumor-like spheroids. To characterize the tumor-like spheroids formed by MIA cells and PSCs, the cells were labeled by Fluorescence Cell Linker Kits (Sigma-Aldrich, USA) following the manufacturer's instruction. These fluorescent dyes have a long aliphatic tails incorporates into membrane of the cell and have a half-life over 30 days in vitro [30], which can be used for long-term cell tracking [31]. Using the kits, MIA cells and PSCs were re-spectively stained with PKH26 (red color) and PKH67 (green color). After labeling, the cells were washed with 10 ml culture medium twice before seeding on CS-HA coated plates. MIA cells and PSCs were seeded on the CS-HA coated plates in different seeding ratios as indicated in the main text. The morphologies of cells cultured were observed at 24 h and 48 h after seeding by an inverted microscope (Leica, DMIRB). A fluor-escence microscope was employed to examine the structural organiza-tion of tumor spheroids. To determine the size of spheroids, 5–10 images (> 10 spheroids in an image) and approximately 50–70 spheroids were adopted for each group. The size of tumor spheroids was analyzed by the NIS-Elements software (Nikon).
    2.4. Confocal imaging
    Cells subjected to confocal imaging were pre-stained with PKH26 and PKH67 as mentioned above. After 48 h of spheroid formation, the spheroids were fixed for overnight at 4 °C with 4% paraformaldehyde in PBS followed by 2–3 washed with PBS. To better visualize the internal cell arrangement in the co-spheroids formed, EasyIndex (Lifecanvas technologies) solution was used to increase the transparency of the spheroids. Before images were taken, the spheroids were soaked into the EasyIndex. The images of tumor-like spheroids were taken with a Leica TCS SP5 II confocal laser scanning microscope. Z-stack was per-formed with a Z-stack of 5 μm.
    2.5. Cell number counting for spheroids
    To determine the cell number and respective population of tumor-like spheroids formed by MIA cells and PSCs, MIA cells were pre-stained with PKH26 to distinguish them from PSCs in the co-spheroids. The cells cultured on the CS-HA coated plate were collected at 24 h, 48 h, and 72 h. Spheroids were dissociated by Accumax™ (Stem cells, Dayton, Ohio) enzyme. The final volume of the cell suspension was 500 μl and incubated for 30 min at 37 °C. After incubation, the cell number was counted by a cell counter (Countess II FL, Life technologies) with a fluorescence imaging module. The experiments were conducted in-dependently for three times to obtain the average cell number.