# Elucidating the Role of Matrix Porosity and Rigidity in Glioblastoma Type IV Progression
## 4 Results
### MTT Assay  
The proliferation of encapsulated U87-MG cells within both gels was evaluated at different time points, using thiazolyl blue staining (MTT, Sigma-Aldrich), in which absorbance data correlate to cell metabolic activity. In brief, at each time point, the medium was removed and replaced with 1 mL fresh medium supplemented with MTT stock solution at a final concentration of 5 mg/mL. After 3 h incubation, medium was removed and the converted dye was solubilized by the addition of 1 mL ethanol absolute. To obtain a homogenous solution, hydrogels were disrupted mechanically via a potter [29]. Absorbance was measured at 570 nm with background subtraction at 670 nm.  
With time, cells were also observed to change their morphology as they were proliferating and moving about the hydrogels. This is clearly documented by SEM images in Figure 5C,D, respectively, for COL and HA. Initially, U87-MG cells presented a rounded shape on both scaffolds. However, at later time points, cells cultured in COL were mostly elongated presenting a bipolar, spindle-shaped morphology, whereas cells cultured in HA mostly preserved the original rounded shape. Figure 8 shows similar morphologies for U87-MG cells in COL already at day 3. It should also be noted that U87-MG cells in HA have been observed to cluster together forming larger aggregates, differently from cells in COL which are rather isolated. This is already evident at day 3 as evidenced by the right column images representative of higher magnifications of those on the left side (Figure 8). This behavior is also confirmed by the fluorescent images presented in Figure 5E,F. Again, U87-MG cells cultured in COL tend to assume an elongated morphology, which preludes to higher motility and migration potential. Differently, the same cells in HA hydrogels assume a rounded shape and are more prone to clustering as shown in the high magnification images (right column) of Figure 8.  
Figure 8: Cell morphology within the scaffolds. Morphology of U87-MG cells was monitored over time, from day 1 to 7, via scanning electron microscopy. Cells included in COL gels were observed to alter their initial rounded shape into a more spindle-like, elongated morphology. This behavior is already observed at earlier time points. Cells included in HA gels grew into small clusters preserving their original rounded shape. Images on the right column panel of each gel (Collagen type I and Hylavronic acid) represent higher magnifications of the related left ones. Scale bar = 50 μm.  
To further characterize the hydrogel-dependent cell migration behaviors, individual U87-MG GFP\({}^{+}\) cells were monitored by registering their position and measuring the length of the path traced by cell nuclei over time. Tumor cells in COL hydrogels migrated over longer distances as compared to cells in HA, within the same time period. This is reflected by the polar diagrams of Figure 5G,H and in the Supporting Movies S1 and S2. These different cell behaviors are in agreement with previous reports showing that HA favors the formation of cell clusters [14], whereas COL supports more cell spreading [46].