Figure 3
Density dependency of spheroid formation using DEX-in-PEG ATPS pattern.
(A) Mean and standard deviation (n = 3) of densities of PEG- and DEX-rich phases in various concentration ATPSs. As DEX concentration increased, the density of DEX-rich phase increased substantially but the density of the PEG-rich phase increased only slightly. (B) Densities of cell lines used in spheroid formation by PEG/DEX ATPS pattern. Most of the cells were found between 60 to 40% and 40 to 20% Percoll layers. The densities of the Percoll layers were calculated following the manufacturer’s manual. (C) Cell tracker (CMFDA) labeled NIH-3T3 cells were tested for spheroid formation with 6 different PEG/DEX ATPS patterns which covers all the density of 20 to 60% Percoll layers. When the density of DEX-rich phase was less than or similar to the density of the cells, they settled and attached to the culture surface (PEG/DEX 5%/1% to 5%/5%). However, when the density of DEX-rich phase exceeded the density of the cells, they floated, gathered and formed a spheroid at the apex of the DEX-rich pattern (PEG/DEX 5%/7% to 5%/11%). Dotted lines: PEG/DEX interfaces. Scale bar: 500 μm. (D) Stability of spheroids formed using ATPS. The spheroids formed using ATPS were carefully transferred to 30 mm glass-bottom dish. After 3 days, spheroids were generally grown and some of them were merged. The spheroids were then stained with calcein AM and ethidium homo-dimer solution for live/dead analysis. Scale bar: 400 μm for phase-contrast images, 200 μm for fluorescence image (E) The viability of cells cultured with PEG- and DEX-rich phases for 48 h. ATPS (5%/9%) showed almost no cytotoxic effect.
