Bx51tf system microscope

Cells were cultured until passage 2, and 50,000 cells/mL were seeded on a poly-L-lysine-coated cover glass in a 6-well cell culture plate maintained at 37°C in a 5% CO₂ incubator for 2 days in order to perform immunocytochemistry. Briefly, the cells were seeded into a poly-L-lysine-coated cover glass, fixed with ice-cold methanol for 10 min, and washed three times with PBS for 5 min each. Then, they were incubated with 1% BSA in PBS-T buffer for 30 min to block unspecific antibody binding. Cells were then incubated with each primary anti-mouse monoclonal antibody against CD34 diluted at 1 : 500 (Abcam, Cambridge, UK), CD44 diluted at 1 : 500 (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) diluted at 1 : 1000 (Abcam, Cambridge, UK), and CD31 diluted at 1 : 1000 (Abcam, Cambridge, UK) in PBS-T with 1% BSA for 60 min at room temperature. Cells were then washed three times with PBS-T buffer and incubated with the secondary antibody, goat anti-mouse Cy3 ™ (Bethyl Laboratories, TX, USA), diluted at 1 : 500 for 60 min in a dark room to activate and preserve fluorescence. All microscopic images were created on an Olympus® DP71 microscope digital camera installed on an Olympus BX51TF system microscope (Olympus, Tokyo, Japan).

The position of the printed cells was confirmed using Cell Tracker™ (Life Technologies, OR, USA). Epithelial cells were stained with a green dye (Cell Tracker™ CMFDA, Life Technologies, OR, USA), whereas chondrocytes were stained with a red dye (Cell Tracker™ CMTPX, Life Technologies, OR, USA). Microscopic images were obtained with fluorescence imaging using an Olympus^® DP71 microscope digital camera installed on an Olympus® BX51TF system microscope (Olympus, Tokyo, Japan).

Cells were cultured on DMEM-LG growth medium for 4 weeks prior to the assay. The assay was initiated by coating a 24-well cell culture plate (SPL, Gyeonggi-do, Korea) with 150 mL of growth factor-reduced Matrigel (BD Biosciences, NJ, USA) per well. Approximately 50,000 cells were then seeded into each well and incubated at 37°C. After 12 hours later, light microscopic images were taken by the Olympus DP71 microscope digital camera installed on the Olympus BX51TF system microscope (Olympus, Tokyo, Japan).