The stiffness of PA gels can be controlled by adjusting the proportion of acrylamide and bis-acrylamide. It is often chosen to study the effect of substrate stiffness on cell growth and development for its controllability and low cost (Charrier
et al. 2020; Saha
et al. 2010). Substrate stiffness, as an elastic cell growth microenvironment, is currently considered to be an important factor affecting cell behavior (Wells
2008). However, the conditions for neuronal cultivation are harsh, especially for synaptic-formation studies, which require approximately two weeks of primary neuronal cultivation. It is difficult to culture neurons for that long time on PA gel. In this paper, we offered an optimized method of preparing PA gel: making PA gel immediately after the preparation of the amino silicified covering and immediately activating the PA gel (
Fig. 3). Then, the cell adhesion and development of neurons were tested through experiments, and it was found that the optimized method significantly improved the cell adhesion rate and development of primary neurons (
Figs. 4,
5, and
6). In the original method, it was left for a period after the reaction of glutaraldehyde and PA gel. For one thing, we speculate that the glutaraldehyde was easily oxidized to glutaric acid by oxygen due to its reducibility. However, glutaric acid could not react with the subsequent acrylamide, and these excess glutaric acids will generate toxicity to cell adhesion and growth in the later stage. For the other thing, one or two weeks were last after the preparation of PA gel with no activation, of which some molecules may have already failed in the HEPES buffer. The failed molecules could not react with sulfo-SANPAN in the crosslinking step. We have provided an optimized PA gel preparation method that effectively avoids the potential cytotoxicity caused by these defects. Also, we have achieved the cultivation of neurons on PA gel with different stiffness substrates for two weeks (data not shown), enabling them to form synapses effectively. Hope that this method offers convenience to researchers studying the role of substrate stiffness in neurons.