Simultaneous enhancement of cell proliferation and thermally induced harvest efficiency based on temperature-responsive cationic copolymer-grafted microcarriers.

The development of large-scale suspension cell cultures using microcarriers has long been a focus of attention in the fields of pharmacy and biotechnology. Previously, we developed cell-detachable microcarriers based on temperature-responsive poly(N-isopropylacrylamide) (PIPAAm)-grafted beads, on which adhering cells can be noninvasively harvested by only reducing the temperature without the need for proteolytic enzyme treatment. In this study, to improve the cell harvest efficiency from bead surfaces while maintaining cell adhesion and proliferation properties, we prepared temperature-responsive cationic copolymer-grafted beads bearing a copolymer brush consisting of IPAAm, positively charged quaternary amine monomer (3-acrylamidopropyl trimethylammonium chloride; APTAC), and hydrophobic monomer (N-tert-butylacrylamide; tBAAm). The incorporation of positively charged APTAC into the grafted copolymer brush facilitated bead dispersibility in a cell culture system containing Chinese hamster ovary (CHO-K1) cells and consequently allowed for enhanced cell proliferation in the system compared to that of unmodified CMPS and conventional PIPAAm homopolymer-grafted beads. Additionally, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted beads exhibited the most rapid and efficient cell detachment behavior after the temperature was reduced to 20 °C, presumably because the highly hydrated APTAC promoted the overall hydration of the P(IPAAm-co-APTAC-co-tBAAm) chains. Therefore, P(IPAAm-co-APTAC-co-tBAAm) terpolymer-grafted microcarriers are effective in facilitating both cell proliferation and thermally induced cell detachment in a suspension culture system.

The use of biotin-avidin binding to facilitate biomodification of thermoresponsive culture surfaces.

Here, we report biomodification of temperature-responsive culture surfaces with biotinylated biomolecules utilizing streptavidin and biotinylation of the surfaces. Poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) was covalently grafted onto tissue culture polystyrene (TCPS) dishes. Biotinylated Arg-Gly-Asp-Ser (RGDS) peptides with different spacer lengths (biotin-conjugated G(n)RGDS (n=1,6,12,16)) were examined. Human umbilical vein endothelial cells (HUVECs) adhered and were well spread on G(12)RGDS-immobilized surfaces in the absence of serum at 37 degrees C, while much less cell adhesion was observed with the other peptides. Adhered HUVECs were detached on reducing temperature to 20 degrees C, or on adding free RGDS peptide. Interestingly, cell detachment was accelerated by applying both these techniques. Consequently, by optimizing the spacer length, biomolecules can be functionally immobilized onto thermoresponsive surfaces via the affinity binding between avidin and biotin.