T-cell mediated cytotoxicity in lung-cancer-on-chip cultures.

A. Experimental design: the lung cancer IGR-Pub cells are embedded in a collagen matrix in the central chamber of the chip, alone or together with autologous CTLs (P62 clone) at 1:1 effector to target cell (E:T) ratio; cells are live-imaged in transmission channel and fluorescence channels (red and green) every hour for 48 h. B. Representative images of IGR-Pub cells after 1 h, 24 h and 48 h of culture on-chip, alone (uppers panels) or with autologous T cells (lowers panels). Red arrows indicate living cells, whereas green arrows point at apoptotic cells. Blue arrows point at CTLs. Scale bar, 100 μm. C. Time-course quantifications of the apoptosis rate, calculated in 10 h-time-intervals, showing the comparison between manual counts (black rounds) and automatic counts (red squares, T_LAG = 10 h), without (left) or with (right) T cells. The means +/- SEM of 3 measurements on 3 view fields from the same experiment were calculated automatically with STAMP every hour and manually every 10 hours. For statistical analysis, the measurements for each of the two conditions (manual and automated) were assembled regardless of the time variable. The non-parametric Mann-Whitney test was applied, since the data did not pass the Shapiro-Wilk normality test, and the difference resulted not significant.