## 3 Results and Discussion
However, when this was attempted, it was found that the levels of DMSO were nearly 100 times the minimum levels for severe cytotoxicity. With regards to washing, most papers describe washing the glycated gels with deionized water; for example, [PERSON] et al., (2014) reported no significant difference in cell viability among scaffolds [7]. As others reported [7], we do not believe that the use of small amounts of genipin to cross-link collagen causes cytotoxicity. One possible reason for cytotoxicity would be the use of DMSO. We believe that washes with only dH2O were not sufficient to eliminate cytotoxicity from the glycated gels. [PERSON] et al., (2008) reported a seven-time decrease in the number of cells on their 10 mM genipin gel when compared to their control. However, their gels were unwashed, and instead of HUVECs, L929 fibroblasts were used [15]. Claudin-5 was stained and imaged under confocal microscope to exam cell morphology (Figure 7). Average relative fluorescence among multiple cells per treatment was measured, and the expression of claudin-5 per scaffold was quantified. Results from Claudin-5 staining show where the intercellular junctional area is located, clearly showing the morphology of cells. It was found that glycation increased the cellular expression of claudin-5 when compared to the control. Even though the control sample (collagen only) shows higher viability (Figure 6), the cells in the control sample show more rounded morphology. Among the glycated scaffolds, the 10, 13, and 22 mM scaffolds had higher claudin-5 expression than the 1 mM gel, but the 10 mM and the 13 mM scaffolds had no significant difference in protein expression (Figure 8). Further cell spreading was analyzed using Image J. As described in Figure 8, pre-glycated gels had a higher degree of cell spreading than in post-glycated or control scaffolds. However, while the 22 mM gels had  
Figure 6: (**A**) Cell proliferation and (**B**) viability: cell counts were measured in three different areas and averaged over the course of 3 days (\(p\) = 0.9942), and live cells were stained with Calcein-AM and quantified in ImageJ (\(p\) = 0.2213). significantly higher levels of cell spreading from all other gels, the 13 mM scaffolds were found to have no significant difference from both the 1 and 10 mM gels. **TEER**: As shown in Figure 9, the increase of genipin concentration increased TEER resistance. In particular, the 10, 13, and 22 mM scaffolds had significantly higher resistance than the bare Transwell membrane, as well as the control and 1 mM scaffolds. Figure 8: **(A)** Claudin-5 expression and **(B)** average cell area of HUVECs grown on control, pre- and post-glycated gels. Measurements were performed in triplicate on day 3 of HUVEC culture (\(p<0.0001\)). Figure 7: Claudin-5 expression (green) and Hoechst nuclei stain (blue). Letters **(a–e)** refer to control, 1 mM, 10 mM, 13 mM, and 22 mM concentration gels, respectively. Images taken at 10X. Scale bar = 100 μm. TEER measurements resulted in an exponential-like curve in resistance at 3 days of growth with the increase of genipin concentration. There was variability in resistance measurements per sample, because collagen scaffolds are not flat surfaces, and the cells had not formed a perfect monolayer, thereby meaning that cell density varied in certain areas. This increase in TEER indicates the formation of a better endothelial barrier from gels grown on stiffer scaffolds, which is consistent with the other results of endothelial response observed in this experiment. Generation of mechanical tunable matrix is critical for in vitro culture platform development such as tissue chip and organoid, in order to model diseases and disorders such as alzheimer's disease; (1) alternative testing platform development to animal model; and (2) regenerative engineering such as wound healing.