Chemically grafted fibronectin for use in QCM-D cell studies

Abstract

Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using spectroscopic ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics.

Keywords: Biocompatibility; Cell mechanics; Endothelial cells; Fibronectin; Quartz crystal microbalance; Surface coating.

Fig. 1

(a) Experimental scheme of fibronectin immobilization onto silicon oxide surfaces, including glass microscope coverslips, hydroxylated silicon wafers, and silicon oxide coated QCM sensors, using the well-known epoxide-amine reaction. (b)Table displaying ellipsometric thickness and contact angle of dry layers. After ¹30 min and ²12 h exposure of FN solution to SiO₂ surfaces, respectively, contact angle and thickness were measured between 3 and 5 times for each surface.

Fig. 2

(a) Topology and (b) phase AFM images of chemFN and physFN on SiO₂ coated QCM sensors, respectively. The scan area of each image shown is 1 × 1 m². (c) RMS roughness of SiO₂, physFN, and chemFN surfaces. RMS roughness is reported as mean±SD. Roughness values were determined from 25 separate 1 μm² subsections taken from 5 × 5 m² images for each substrate type. ## represents p<0.001 versus physFN, *** represents p<0.0001 versus the SiO₂ surface, with p<0.05 considered to be significantly different.

Fig. 3

(a) Calcein (vital dye) and phalloidin (actin dye) stained cells on chemFN and physFN surfaces. The brightness-contrast of the calcein images was adjusted to show the same range of intensities. All scale bars are 100 μm. Calcein images were taken with a 20× objective lens, and phalloidin images were taken with a 60× oil-immersion objective. (b) ATP stimulation of fluo-4 loaded cells. (i) A group of fluo-4 labeled chemFN cells before and after stimulation with ATP. T=0 is considered to be when the ATP is added to the dish. Scale bar is 20 μm. (ii) Representative traces of the calcium signal of a cell plated on a chemFN surface (black) and a physFN surface (gray). Inset: average fluorescence ratios for all responding cells on chemFN (44/49) and physFN (34/36) surfaces. A Student’s t-test comparing the two groups gave p=0.91.

Fig. 4

(a) Experimental scheme for data collection using QCM-D. Stage I: Collecting frequency (Δf) & dissipation (ΔD) data using chemFN or physFN coated QCM sensors and PBS solution (flow rate=100 μL/min, 21 °C); stage II: collecting Δf & ΔD using the QCM-sensor from stage I with cells under the same conditions as stage I; stage III: collecting Δf & ΔD using the cleaned SiO₂-coated QCM sensor with the same conditions as stage I. (b) Overall combined traces of (i) Δf_n/n (n=3, 5, 7) and (ii) ΔD_n of a cleaned SiO₂-coated QCM sensor (stage III), the fibronectin layer on a SiO₂-coated QCM sensor (stage I), and the cell adherent layer on representative physFN and chemFN QCM-D sensors (stage II) in PBS. The data from stages I, II, and III were stitched together for modeling in the order III-I-II using Q-soft (Q-Sense). Simulated and experimental curves for Δf_n/n (n=3, 5, 7) and ΔD_n vs. time show a good fit between the viscoelastic model and the experimental data. (iii) Thickness of the fibronectin and cell layers in PBS as determined from the fits shown in (i) and (ii). (iv) Viscosity and shear modulus of the fibronectin and cell layers as determined from the fits shown in (i) and (ii).

Fig. 5

(a) Representative fluorescent images of (i) low cell density (753 cells/cm²) and (ii) high cell density (24,448 cells/cm²) on chemFN QCM sensors, respectively. Both scale bars are 200 μm. The image shown in (i) was taken at 4×, while the image shown in (ii) was taken at 10×. (b) Viscosity (black) and shear modulus (gray) versus thickness of the cell layer on the chemFN layer. Exp 1 corresponds to (a(i)) and exp 2 corresponds to (a(ii)). The properties of the chemFN layer (derived from exp 1) are as follows: in-situ thickness=6 nm, viscosity=1.64 × 10⁻³ Ns/m², shear modulus=0.45 × 10⁴ N/m².

Fig. 6

(a) Real-time frequency and dissipation changes of a chemFN coated crystal plated with fibroblasts and then subjected to 1 μM cytD. (b) Fibroblasts stained with phalloidin to highlight the actin cytoskeleton. (i) shows control cells on a crystal treated with 0.1% DMSO only, while (ii) shows cells on the crystal treated with cytD. The scale bar is 20 μm.