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

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.

Grafting versus Crosslinking of Silk Fibroin-g-PNIPAM via Tyrosine-NIPAM Bridges

“…Thus, the silk fibroin spectra showed four secondary C 1s peaks: a first peak centered at about 284.51 eV, corresponding to C-C; a second peak centered at 285.25 eV, which can be assigned to C-H (CH 3 in alanine; -CH 2 - and -CH- in backbone); a third peak centered at 286.12 eV, which can be assigned to C-O/C-N; and a fourth peak centered at 287.96 eV, which can be assigned to C=O/N-C=O [34]. The C 1s spectra of PNIPAM revealed four peaks: a first peak centered at about 284.97 eV, assigned to C-C contribution, and three deconvoluted peaks centered at 285.92, 287.05 and 288.16 eV, assigned to multiple C-H groups, C-O/C- and C=O/N-C=O, respectively [35,36]. The C 1s spectra of SF-g-PNIPAM (0.1 w / w to SF) also revealed four peaks shifted to lower values with respect to the silk fibroin spectrum: the first peak was centered at about 283.98 eV, which can be assigned to C-C, with the contribution of a new carbon species from PNIPAM structures [35,36].…”

Cell sheet tissue engineering: Cell sheet preparation, harvesting/manipulation, and transplantation

“…4(C), lower]. Previous reports indicated that no fibronectin was released from PIPAAm or heparin-immobilized poly(IPAAm-co-CIPAAm) surfaces on which cells were not cultured after reducing temperature to 20 C. 37,52 These imply that the pulling force that is generated by the contraction of stress fibers is essential for the detachment of fibronectin and bFGF, which are bound with receptors through affinity (e.g., K D between a fibronectin fragment and an integrin α 5 β 1 , 4.4 nM 53 and that between bFGF and FGFR1, 61.9 nM). 49 HB-EGF-bound heparin-immobilized temperature-responsive surfaces facilitate the fabrication of hepatocyte sheets with preserved hepatic functions.…”

Thermoresponsive Poly(glycidyl ether) Brush Coatings on Various Tissue Culture Substrates—How Block Copolymer Design and Substrate Material Govern Self-Assembly and Phase Transition

“…Coatings based on thermoresponsive polymers consistently prove to be a valuable foundation for cell sheet engineering purposes [ 1 , 2 , 3 ]. Due to their physical response to changes in temperature under aqueous conditions, such coatings can switch from a rather hydrophobic, protein- and cell-adhesive state into a more hydrophilic, protein- and cell-repellant state upon cooling [ 4 , 5 ]. This phase transition is characterized by an increase in coating hydration, which is usually accompanied by the swelling of the thermoresponsive polymer layer.…”