A ready‐to‐use fast gelation/liquefying hydrogel towards enzyme free three‐dimensional cell culture

Trypsin treatment employed in traditional cell desorption method cause the damage of extracellular matrix protein and cell membrane integrity. Two-dimensional thermo-sensitive cell culture dish has the limitation of insufficient area. This report describes an ingenious design and synthesis of a thermoresponsive chitosan-based hydrogel, N-succinyl chitosan (NSCS) grafted poly(N-isopropylacrylamide) (PNIPAM), named NSCS-g-PNIPAM. We then demonstrate the ultrafast gelation and liquefying of NSCS-g-PNIPAM by reversibly tuning the temperature of hydrogel solution above (gelation) or below (liquefying) 32 degrees C. These unique features render the NSCS-g-PNIPAM hydrogel an ideal matrix for enzyme-free three-dimensional cell culture. As an exemplar, the skeleton muscle cell is chosen for in vitro cellular studies. The data suggests that after seven passages and harvesting, the amount of total protein, laminin and fibronectin of cell collected from the thermoreponsive hydrogel group shows unsignificant decrease after seven passages. The proteins on the cell surface are well protected compared to the conventional trypsin-treated method. In addition, by labeling the NSCS-g-PNIPAM hydrogels with tetraphenylethylene (TPE)-based aggregation-induced-emission (AIE) luminogenes, NSCS-g-PNIPAM were observed by confocal microscopy and they remain outside during entire cell culturing period.