Morphological analysis of the tumor spheroids.
The progressively developing spheroids were observed at 24 h intervals. The morphological characteristics of spheroids including their diameter and shape were measured by optical analysis using a EVOS XL Cell Imaging System microscope (Thermo Fisher, Waltham, MA USA). The mean diameter of the 3D structures was calculated by using ImageJ Software (1.48v).
For SEM imaging the tumor spheroids embedded within the hydrogels were fixed with glutaraldehyde (2.5%) in cacodylate buffer (0.1 M) at 4 °C O.N. The fixed specimens were washed three times with PBS, and 1% osmium tetroxide in a cacodylate buffer was added for 6 h. Then, the samples were washed three times with PBS, cut and lyophilized. Finally, the as-prepared samples were transferred to the SEM microscope to be imaged. The operating conditions of the microscope were the same as those used for imaging the hydrogels.