*2.7. Cytotoxicity Test*

The cytotoxicity test of hydrogels was performed as previous method using U87-MG glioblastoma cells (ATCC HTB-14) cultured by Eagle's Minimum Essential Medium (EMEM) with 10% fetal bovine serum (FBS) and 100 IU ml−<sup>1</sup> penicillin and streptomycin [13,36]. The hydrogels were soaked in DI water for 8 days, followed by being immersed in the cell cultural medium for 3 days to remove residual monomer and chemicals. After that, the washed hydrogels were put into another cell cultural medium again for 3 days to achieve conditioned cell cultural medium. Following this, 2 × 10<sup>4</sup> U87-MG cells were seeded onto a 24-well plate and cultured in fresh cell cultural for 3 days. Then the conditioned cell cultural medium was used to culture the cells for another 24 h or 48 h. To evaluate the cytotoxicity of hydrogels, a crystal violet staining method was used. First, cells were fixed by 1 mL of 2% glutaraldehyde in triplicate for 10 min. Then the medium was aspirated and the cells were washed with phosphate-bu ffered saline (PBS) twice. Then, 1 mL of 0.1% crystal violet was added into the plate and the cells were stained for 40 min. Crystal violet was then washed away by water. Crystal violet was present only in the stained cells. The plate was further drained inversely overnight. The cytotoxicity was evaluated by reading the absorbance at 590 nm of the crystal violet.

In addition, Trypan blue was used to show the cell viability by the previous method [13,36]. Specifically, old media in the 24 well plate were aspirated and 1 mL of Dulbecco's phosphate-bu ffered saline (DPBS) was added to wash the adherent cells. Then, DPBS was added into the counting chamber, and the chamber was inserted into a Cellometer Vision Image Cytometry for counting and imaging.
