*2.2. Cell Viability*

As shown by the results of the viability assay of L929 cells in the presence of CS-GT (Figure 2), cell viability decreased gradually with the increase of CS-GT concentration. After CS-GT and L929 cells were co-incubated for 1 and 2 days, CS-GT at a concentration of 100 μg/mL promoted cell growth to some extent, and cell viability values were 112.65% and 112.23%, respectively, indicating that CS-GT at this concentration is non-toxic to L929 cells. After CS-GT and L929 cells were co-incubated for 3 days, cell viability decreased to 90.79%, indicating that CS-GT at this concentration exhibited limited cell inhibition, possibly because cell proliferation via cell division led to higher cell density and competition among cells for nutrition (*p* < 0.05 compared to days 1 and 2) [33]. For CS-GT at a concentration of 200 μg/mL, cell viability values on days 1 and 2 were 101.68% and 100.44%, respectively, and no significant impact on cell growth was noted. On day 3, cell viability decreased to 86.73%, probably due to combined action of CS-GT and cell division and proliferation (*p* < 0.05 compared to days 1 and 2). For CS-GT samples at concentrations between 300 and 1200 μg/mL, cell viability values on days 1 and 2 decreased in turn, respectively. However, at the same concentration, cell viability decreased less. On day 3, reduction in cell viability at each concentration was greater than those on days 1 and 2. At a concentration of 1200 μg/mL, cell viability decreased to 75.00%, the lowest one among cell viability values at all concentrations. According to ISO standard, after full action between a substance and cells, a concentration with cell viability above 75% is deemed not cytotoxic [34]. In summary, CS-GT samples at concentrations below 1200 μg/mL were non-toxic to L929 cells. In particular, at lower concentrations (e.g., 100 μg/mL) with action time not longer than 2 days, CS-GT promoted cell growth to some extent, indicating CS-GT has good cytocompatibility.

**Figure 2.** Cell viability of CS-GT (mean ± SD, \* *p* < 0.05, *n* = 6).

L929 cells were treated with GT and CS-GT samples at a concentration of 100 μg/mL for 1, 2, and 3 days, respectively, and such cells were stained with AM/PI in these 3 consecutive days to observe the cell state (Figure 3). After reacting with cellular lactonase, calcein-AM emits green fluorescence and renders viable cells stained. Ethidium homodimer-1 binds to the DNA of dead or damaged cells, rendering dead cells stained red. Based on live/dead staining analysis, with the increase of culture time, a grea<sup>t</sup> number of normally-shaped green viable cells and a sequentially increased minor amount of red dead cells were observed in the control group. After L929 cells were treated with GT, more and more red dead cells were observed with the increase in incubation time. While the cells treated with CS-GT were basically normal in morphology, fewer red dead cells were observed, and the variability of cell staining versus time was similar to the case of the control group. Results of L929 cell viability (Figure 2) and observation of cell staining state further indicate that CS-GT is non-cytotoxic to L929 cells and has good cytocompatibility.

**Figure 3.** Live/dead staining analysis of cell compatibility of the sample. L929 cells in GT and CS-GT were stained after incubation for 1, 2, and 3 days.
