Preparation of thermosensitive PNIPAm- based copolymers with tertiary amino and hydroxyl monomer for cell attachment and detachment study

Thermo-responsive microcarriers are essential for large-scale expansion of anchorage-dependent cells in a suspension culture. In this study, the authors prepared four different N-isopropylacrylamide (NIPAm)-based thermosensitive copolymers by copolymerizing with N-hydroxyethyl acrylamide (HEAA), 2-Hydroxyethyl methacrylate (HEMA), 2-(Diethylamino) ethylmethacrylate (DEAEMA), and 2-(Dimethylamino) ethylmethacylate (DMAEMA) through free radical polymerization (FRP) and used them for surface modification of Cytodex-3 microcarrier. The thermosensitive characteristic of the copolymers enabled microcarriers to attach and detach cells in response to temperature changes. Herein, NIH 3T3 cells were used as a model cell line and it was found that the cells' adhesion rate on Cytodex-3 microcarriers modified with different formulations of thermosensitive copolymers was greater than unmodified microcarriers. The microcarriers coated with 5 wt% of the copolymer showed better cell attachment than 3 wt% and 10 wt% copolymer coated microcarriers. Particularly, 5 wt% P(NIPAm-5% HEMA) copolymer coated microcarriers showed excellent cell attachment than other formulations. More importantly, as the temperature was reduced to 4 °C, the cells cultured on copolymer modified microcarriers were completely detached as a cell sheet from it without proteolytic enzyme treatment. Besides preserving the functionality and integrity of cellular structures, the thermal-induced detachment of cells from modified microcarriers was much higher (∼9.9 x 104 cells/mL) than cells detached from the original Cytodex-3 (∼7.5x103 cells/mL) in 1 h at 4 °C. Therefore, this study verified that microcarriers modified with thermosensitive copolymers exhibited enhanced cell attachment and detachment with temperature change, while maintaining cell functionality, thereby providing adequate number of viable cells for large scale production.