Simultaneous Enhancement of Cell Proliferation and Thermally Induced Harvest Efficiency Based on Temperature-Responsive Cationic Copolymer-Grafted Microcarriers

Abstract

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.

PNIPAM grafted surfaces through ATRP and RAFT polymerization: Chemistry and bioadhesion

“…As cell membranes are anionic, cationic surfaces have to be carefully used since they can damage cells and tissues, whereas negatively charged polymers will electrostatically repel cells [18][19][20][21]. The importance of surface/cell interactions has been investigated for many years, and an interesting discussion was published in 2001 by Castner et al [17].…”

Engineering dynamic biointerfaces

“…Dynamic chemical [13–17], optical, thermal [18] or electrical [19–21] cues modulate cell-adhesion characteristics of substrates. Mimicking the in-vitro behavior of heterotypic cellular contacts requires micrometer-scale control over cell attachment and spreading, imposing limitations on the technique.…”

Thermosensitive PHEMA microcarriers: ATRP synthesis, characterization, and usabilities in cell cultures

“…The grafted amount of PNIPAAm (m g ) onto the PHEMA microbeads was determined from the nitrogen content increase obtained from the CHNS elemental analyzer (Vario-MICRO) by using the Equation (1) given below [17]:…”

“…There are several studies showing the successful grafting of thermally responsive PNIPAAm brushes onto various polymeric particles by ATRP. [15][16][17]25,26] In our study, by taking into account the several advantages of ATRP and due to the nontoxicity and biocompatibility of PHEMA, thermoresponsive microcarriers were produced by grafting PNIPAAm onto hydroxyl groups carrying crosslinked PHEMA beads. Grafting of PNIPAAm by ATRP synthesis was carried out in two subsequent steps: firstly, an ATRP initiator, 2-bromopropionyl 6 M. Gümüşderelioğlu et al…”

“…[16] Moreover, they improved cell harvesting efficiency of those beads by grafting terpolymer brushes, including PNIPAAm. [17] Cell culture studies performed on PNIPAAm-grafted thermoresponsive surfaces indicated that the removal of mammalian cells via low-temperature detachment from these surfaces was less destructive to the extracellular matrix (ECM) proteins than enzymatic digestion. In light of such information, although commercial thermosensitive Petri dishes are produced by Nunc for specialized cell culture studies, a commercial thermosensitive microcarrier is not available yet.…”