Effect of Temperature Changes on Serum Protein Adsorption on Thermoresponsive Cell-Culture Surfaces Monitored by A Quartz Crystal Microbalance with Dissipation.

Jun Kobayashi,Nobuhiko Yui,Yoshikatsu Akiyama,Teruo Okano,Yoshinori Arisaka,Masayuki Yamato

doi:10.3390/ijms19051516

Abstract

Thermoresponsive cell-culture polystyrene (PS) surfaces that are grafted with poly(N-isopropylacrylamide) (PIPAAm) facilitate the cultivation of cells at 37 °C and the detachment of cultured cells as a sheet with an underlying extracellular matrix (ECM) by reducing the temperature. However, the ECM and cell detachment mechanisms are still unclear because the detachment of cells from thermoresponsive surfaces is governed by complex interactions among the cells/ECM/surface. To explore the dynamic behavior of serum protein adsorption/desorption, thermoresponsive surfaces that correspond to thermoresponsive tissue-culture PS dishes were formed on sensor chips for quartz crystal microbalance with dissipation (QCM-D) measurements. X-ray photoelectron spectroscopy (XPS) measurements and temperature-dependent frequency and dissipation shifts, Δf and ΔD, using QCM-D revealed that the thermoresponsive polymers were successfully grafted onto oxidized, thin PS films on the surfaces of the sensor chips. Increased amounts of adsorbed bovine serum albumin (BSA) and fibronectin (FN) were observed on the thermoresponsive polymer-grafted surfaces at 37 °C when compared with those at 20 °C because of enhanced hydrophobic interactions with the hydrophobic, thermoresponsive surface. While the calculated masses of adsorbed BSA and FN using QCM-D were 3–5 times more than those that were obtained from radiolabeling, the values were utilized for relative comparisons among the same substrate. More importantly, the thermoresponsive, dynamic behavior of serum protein adsorption/desorption was monitored using the QCM-D technique. Observations of this dynamic behavior revealed that the BSA and FN that were adsorbed at 37 °C remained on both surfaces after decreasing the temperature to 20 °C.

Highlights

Physisorption of biomolecules and cells onto the surfaces of biomaterials occurs irreversibly through nonspecific interactions
Thermoresponsive polymer-grafted PS surfaces on sensor chips were prepared by spin coating PS and covalently grafting the thermoresponsive polymer by electron-beam irradiation (Figure 1)
Thermoresponsive surfaces were formed on PS-coated sensor chips to replicate thermoresponsive tissue-culture PS dishes

Summary

Introduction

Physisorption of biomolecules and cells onto the surfaces of biomaterials occurs irreversibly through nonspecific interactions. Our laboratory covalently introduced a thermoresponsive polymer, poly(N-isopropylacrylamide) (PIPAAm), onto surfaces to regulate the interactions with biomolecules and cells by temperature changes [1,2]. Tissue-culture polystyrene (PS) surfaces that were grafted with PIPAAm by electron-beam irradiation enable attached cells to be reversibly detached [4,5]. Based on this technology, a thermoresponsive cell-culture dish has been produced and is commercially available as UpCell®. When the temperature was lowered to 20 ◦C, the surfaces became hydrophilic, according to hydration of the grafted PIPAAm, and a confluently cultured cell monolayer was detached as a single cell sheet. Layered cell sheets form three-dimensional tissues [7] and are transplantable to treat damaged tissues and organs [8,9,10,11,12,13]

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