New aspects of chitosan in cell culture: focusing on the pH-responsive characteristic in the mammalian cell culture range

Chitosan, the major derived from second abundant nature polymer chitin, has many biomedical applications because of its biocompatibility, biodegradability, and low toxicity. Structurally, it is a nature cationic polysaccharide composed randomly distribution of N-glucosamine and N-acetyl-glucosamine groups. The presence of primary amine of glucosamine residue makes chitosan a pH-responsive polycation and has potential to apply in pH-responsive aspect, such as drug delivery, tissue-engineering, or related medical device. However, this pH-responsibility has never been applied and discussed for a cell culture system. In this thesis, we developed a culture system based on the pH-responsive nature of chitosan which can control the cell-substrate interaction by precise adjustment of medium pH. We discovered that pH-responsive chitosan was able to control cell behavior in response to small changes in environmental pH, which was at useful pH suitable for recovering cultured cells without additional enzymatic treatment and extensive washing steps. The relationship of cell attachment/detachment, serum fibronectin adsorption, and medium pH was illustrated. Furthermore, a time and cost effective, simple-operative, and wide-extensive platform based on chitosan was developed, based on the different cell detachment ratio from chitosan when raising medium pH. The different detachment ratio was found to be correlated with cell shape. By artificial mixing different detachment ratio of cells, the application of this system was revealed. The importance of this system for primary renal proximal tubular epithelial culture was also demonstrated. Moreover, the role of intracellular cytoskeleton in this pH-induced detachment from chitosan surface was further investigated. The importance of Rho/ROCK-related cytoskeletal action was illustrated. In addition, the reason of HaCaT restricted by this pH-induced detachment on chitosan was investigated. The role of β4-integrin and its ligand in the restriction of pH-induced detachment from chitosan was discussed. Finally, the future perspective of this pH-responsive chitosan culture system was demonstrated.