Development of a rapid in vitro tissue deadhesion system using the thermoresponsive sol-gel transition of hydroxybutyl chitosan.

In regenerative medicine, it has become increasingly important to collect cultured tissues using non-invasive methods. Enzymatic deadhesion is normally used to collect cells, but this method cannot be used when trying to collect whole tissues in order to avoid damaging cell-cell interactions. In order to resolve this issue, a thermoresponsive culture dish with poly(N-isopropyl acrylamide) (PNIPAAm) has been employed. This system can change its hydrophobicity depending on temperature. Thus, tissues can attach above 37 °C and be detached below 20 °C. However, the PNIPAAm system has some issues related to cost and detachment time. In this study, we developed a novel thermoresponsive detachment system using a polysaccharide derivative. We chose hydroxy butyl chitosan (HBC) as the thermoresponsive polymer because of its high biocompatibility and rapid phase transition. We developed a novel method of HBC synthesis in conditions that were milder than previously reported. We used spin-coating to make a thin coating on two kinds of culture dishes with various concentrations of HBC solution. Seeded cells attached to the surface at less than 0.5 mg/ml HBC coating concentration, and they could be successfully detached by simply lowering the temperature of the suspension dishes without enzymatic treatment; the cells took only 5-20 min to detach. To evaluate this system, we measured three metrics related to cell culturing on culture dishes: initial attachment rate, detachment rate and tissue detachment time. The study revealed that tissues could be detached faster on the suspension dishes used in this study than on PNIPAAm grafted dishes when HBC was coated at 0.5 mg/ml. We successfully developed a novel tissue detachment system using HBC. These results suggest that smart polymers may be useful in regenerative medicine.