Thermoresponsive surface prepared using thermoresponsive polymers with low critical solution temperatures (LCSTs) is attractive candidates for stem cell culture because stem cells can be detached from the surface without applying an enzymatic digestion method and, instead, by decreasing the temperature, which enables cell aggregates or cell sheets to be obtained in culture medium. In this study, we designed the thermoresponsive nano-brush surfaces for human stem cell culture (human adipose-derived stem cells [hADSCs] and human pluripotent stem cells [hPSCs]). Using RAFT polymerization, we prepared the coating copolymers having polystyrene and (a) thermoresponsive poly(N-isopropyl acrylamide), PNIPAAm[1], (b) biocompatible polyethylene glycol methacrylate (PEGMA), and (c) polyacrylic acid (PAA) where bioactive oligopeptide (oligo-vitronectin)[2],[3] can be conjugated via carboxylic acid of PAA. hADSCs were cultured on the surface coated with copolymers containing PNIPAAm, PEGMA and PAA conjugated with oligo-vitronectin.
The optimal surface composition where hADSCs can attach and detach by decreasing temperature was investigated. Furthermore, human embryonic stem cells (WA09) and human induced pluripotent stem cells were cultured on the surface coated with these copolymers. We have successfully cultured human stem cells on the thermoresponsive nanobrush surface and can reach 80% cell detachment of hADSCs and 50% cell detachment of hESCs.
However, if we could not reach 100% detachment of stem cells, we have another option that thermoresponsive nanobrush surfaces can be reused with partial detachment of stem cells, which do not need the process to reseed cells (passage). We also investigated whether hPSCs could maintain their pluripotency on the surface coated with these copolymers to find out long period (>passage ten) and whether hPSCs could be easily detach from the surface by decreasing the temperature. This results indicate that human stem cells which are highly sensitive to enzymatic treatment can be achieved by used copolymer nanobrush surfaces. Furthermore, we are designing to shift to a novel 3D culture system to scale up for clinical application.
Akon Higuchi; Yen Ming Chen; Hong Ren Lin; Saradaprasan Muduli; I Chia Peng; Hsing Fen Li
References:
[1] J. Kobayashi, et al., Surface design of antibody-immobilized thermoresponsive cell culturedishes for recovering intact cells by low-temperature treatment, J Biomed Mater Res Part A, 102A, 3883–3893 ( 2014)
[2] A. Higuchi, et al., Physical Cues of Biomaterials Guide Stem Cell Differentiation Fate, Chem. Rev., 113, 3297−3328 (2013)
[3] A. Higuchi, et al., Design of polymeric materials for culturing human pluripotent stem cells: Progress toward feeder-free and xeno-free culturing, Prog. Polym. Sci., 39, 1348-1374 (2014)