Introduction: In regenerative medicine, an effective cell separation method that can provide adequate purity, yield, and function have been needed for effective fabrication of transplantable tissues. As new cell separation tool, we prepared thermoresponsive cationic copolymer brush, poly(N-isopropylacrylamide(IPAAm)-co-N,N-dimethylaminopropyl acrylamide(DMAPAAm)-co-N-tert-butylacrylamide (tBAAm)) brush grafted surfaces through surface-initiated atom transfer radical polymerization (ATRP). Adhesion and detachment behaviour of human bone marrow mesenchymal stem cell (hbmMSC) and other human bone marrow derived cells on the ionic copolymer brush was observed for investigating the possibility of the prepared copolymer brush as a stem cell purifying materials. Experimental: An ATRP initiator, 2-(m/p-chloromethylphenyl) ethyltrimethoxysilane, was modified on glass coverslips (Fig. 1). Copolymerization of IPAAm (75mol%), DMAPAAm(5mol%), and tBAAm(20mol%) was performed through surface-initiated ATRP for 16 h at 25°C using CuCI/Me_6TREN as a catalyst and 2-propanol as reaction solvent. In the polymerization, a-chloro-p-xylene was added as a free ATRP-initiator and brush length was estimated using the synthesized copolymer in the reaction solution. Cell adhesion and detachment behaviour of hbmMSC and other human bone marrow derived cells were observed at 37 and 20°C. Results and Discussion: Prepared thermoresponsive cationic copolymer brush was characterized by ATR/FT-IR, GPC measurement, and zeta potential measurement. As result, amount of grafted copolymer and graft density were 1.51 mg/cm~2 and 1.04 chainsm~2, respectively. Zeta potential of copolymer brush was 19.8 mV. These result indicated that dense thermoresponsive cationic copolymer brush was formed on glass substrate. Cells adhesion and detachment properties on copolymer brush were observed using hbmMSC and other bone marrow derived cells. hbmMSC adhered on the cationic copolymer brush at 37 °C, while other bone marrow derived cells did not. With reducing temperature to 20 °C, adhered hbmMSC detached from cationic copolymer brush. Using this properties, purification of hbmMSC was performed simply by changing temperature. Mixture of human bone marrow derived cells were seeded on cationic copolymer brush. Only hbmMSC adhered on cationic copolymer brush at 37°C. Then, temperature was reduced to 20 °C, and purified hbmMSC was successfully recovered from the copolymer brush (Fig.2). The result indicated that prepared thermoresponsive cationic copolymer brush would be useful for purification of hbmMSC from other cells. Conclusion: Thermoresponsive cationic copolymer brush prepared through surface-initiated ATRP can selectively adhered and detach hbmMSC by external temperature change. Thus, the thermoresponsive cationic copolymer brush would be useful for purification of hbmMSC simply by changing temperature.
展开▼
