Green dio

To visualize the mutual EV uptake, cells were first dyed with fluorescent, lipophilic carbocyanine tracers, green DiO (ThermoFisher Scientific, Waltham, MA, USA, catalog #D3898), and red DiI (ThermoFisher Scientific, catalog #D3886). Right before seeding, cells were treated with 1 uM dye followed by incubation at 37 °C for 5 min [33 (link)]. The GBM was dyed red with DiI, whereas the NSCs were dyed green with DiO. EVs secreted by each cell line take the color of the dye of the parent cell. GBM and NSCs were independently cultured for 24 h in a 6-well non-tissue culture treated plate in 4 mL of culture media, and the cells were incubated at 37 °C with 5% CO₂. The next day, the cells and the conditioned media from each cell line were centrifuged at 0.3 rcf for 5 min at room temperature. The GBM cell pellet was resuspended in NSC-conditioned media, whereas the NSC pellet was resuspended in the GBM-conditioned media with further incubation under the same conditions. The controls did not receive the media switch. After 48 h, the cells were ready to be fixed and visualized using confocal microscopy.

Formation of clear and continuous interface between regions containing different cell types in a contiguous tissue construct was shown in both axial and radial configurations. MCF-7 cells were stained with either green DiO or red DiI fluorescent cell trackers (ThermoFisher). For the axial configuration, half of the tubing was filled with the bioink containing green stained cells (1:3 CMR, 2 × 10⁶ cells/mL solution). After half hour incubation when the collagen had gelled but the cells had not attached to the ECM to apply significant traction forces, the other half of the tubing was filled with the same bioink but with red stained cells. For radial configuration, the whole tubing was filled with green stained cells' bioink (1:3 CMR, 2 × 10⁶ cells/mL solution). After 2 h of incubation that shrinkage was performed, extra medium was extracted, and a 1:3 CMR bioink with 1 × 10⁶ cells/mL was added followed by further incubation. Fluorescent images were taken before and after addition of each bioink using a ChemiDoc™ MP imaging system (Bio-Rad).

To monitor the position of cells seeded using the two-cell insert, primary hippocampal neurons were fluorescently labeled with either DiO (green) or DiL (red) lipophilic tracer dyes (Invitrogen) using standard protocols prior to seeding. One cell type was used for the analysis to avoid confounding variables. Once labeled, the cells were deposited through openings in the insert leading to the inner and outer region of the MEA, respectively. Devices were imaged on DIV 1 (immediately after insert removal), DIV 2, DIV 7, and DIV 22 to record distribution of the fluorescent cells. Images were analyzed using cellSens Dimension (Olympus). Fluorescence intensity was quantified for the inner and outer regions of the electrode array. For each day, fluorescence for the inner and outer regions was calculated as a percentage of total fluorescence (inner and outer regions). Three replicate devices were used for these analyses. Data is represented as mean ± standard deviation. P values were calculated using an unpaired t-test. Data were considered statistically significant for p values < 0.05.