Temperature-modulated separation of vascular cells using thermoresponsive-anionic block copolymer-modified glass

Tadashi Hirotani; Kenichi Nagase

doi:10.1016/j.reth.2024.03.009

Temperature-modulated separation of vascular cells using thermoresponsive-anionic block copolymer-modified glass

Regen Ther. 2024 Apr 6:27:259-267. doi: 10.1016/j.reth.2024.03.009. eCollection 2024 Dec.

Authors

Tadashi Hirotani¹, Kenichi Nagase^{1

2}

Affiliations

¹ Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo, 105-8512, Japan.
² Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.

Abstract

Introduction: Vascular tissue engineering is a key technology in the field of regenerative medicine. In tissue engineering, the separation of vascular cells without cell modification is required, as cell modifications affect the intrinsic properties of the cells. In this study, we have developed an effective method for separating vascular cells without cell modification, using a thermoresponsive anionic block copolymer.

Methods: A thermoresponsive anionic block copolymer, poly(acrylic acid)-b-poly(N-isopropylacryl-amide) (PAAc-b-PNIPAAm), with various PNIPAAm segment lengths, was prepared in two steps: atom transfer radical polymerization and subsequent deprotection. Normal human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts, and human aortic smooth muscle cells (SMCs) were seeded onto the prepared thermoresponsive anionic block copolymer brush-modified glass. The adhesion behavior of cells on the copolymer brush was observed at 37 °C and 20 °C.

Results: A thermoresponsive anionic block copolymer, poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PAAc-b-PNIPAAm), with various PNIPAAm segment lengths was prepared. The prepared copolymer-modified glass exhibited anionic properties attributed to the bottom PAAc segment of the copolymer brush. On the PAAc-b-PNIPAAm, which had a moderate PNIPAAm length, a high adhesion ratio of HUVECs and low adhesion ratio of SMCs were observed at 37 °C. By reducing temperature from 37 °C to 20 °C, the adhered HUVECs were detached, whereas the SMCs maintained adhesion, leading to the recovery of purified HUVECs by changing the temperature.

Conclusions: The prepared thermoresponsive anionic copolymer-modified glass could be used to separate HUVECs and SMCs by changing the temperature without modifying the cell surface. Therefore, the developed cell separation method will be useful for vascular tissue engineering.

Keywords: Cell separation; Endothelial cells; Smooth muscle cells; Thermoresponsive polymer; Vascular tissue engineering.