Cell-repellent polyampholyte for conformal coating on microstructures

Abstract

Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50-150 °C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.

Figure 1

Coating with prevelex and its use for cell aggregate culture. (a) Chemical structure of polyampholyte “prevelex” consisting of phosphate units and amine units. Thin polymer films are conformally formed on substrates via simple coating, drying, and washing processes, altering the surfaces to be highly hydrophilic and electrically neutral. (b) Prevelex-coated surface for resistance to protein and cell adhesion. (c) Spheroids formed in microwell array with conformal prevelex coating.

Figure 2

Characterizations of prevelex-coated surfaces. (a) Water contact angles with and without prevelex coating on PS, glass, and PDMS substrates under aqueous and dry conditions. The bubble contact angle θ_air is measured with a 2.0 µL air bubble in PBS. The water contact angle θ_water is measured with a 2.0 µL pure water droplet in air. θ_air is converted to θ_water by 180 − θ_air. (b) Zeta potential of polystyrene substrates with and without prevelex coating. (c) Dependence of prebake temperature on protein adsorption. QCM sensor is treated with prevelex and heated at the indicated temperature for 1 h. QCM is used to quantify adsorption of molecules in Eagle’s basal medium supplemented with 10% FBS. (d) Protein adsorption after gamma irradiation. The values are presented relative to polystyrene (PS) substrate. (e) Thermogravimetric and differential thermal analysis (TG–DTA) of prevelex. Error bars in (a), (b), and (d) represent the standard error calculated from three independent measurements. Numerical variables are statistically evaluated using a student’s t-test, and *p < 0.05 is considered statistically significant.

Figure 3

Cell repellent of prevelex-coated surfaces. (a) Phase-contrast microscopic images of culture surface 4 days after cell seeding. PS substrates are coated with the different coating agents. (b) Coating thickness and attached cell number. Thickness of coating layer was quantified with spectroscopic ellipsometry. Number of attached cells are quantified by measuring the amount of adenosine triphosphate. Error bars represent standard error calculated from three independent measurements. Numerical variables are statistically evaluated using ANOVA, and *p < 0.05 is considered statistically significant.

Figure 4

Prevelex coating to microstructures. (a) Scanning electron microscopic images of prevelex and 3.6% pHEMA layers coated on microstructured silicon substrates. (b) Coating on sub-micrometer stepped surfaces. Height of the step is 200 nm. (c) Comparisons of coating agents on microwell array plates. PS and PDMS plates are coated with three different coating agents. Cells are observed four days after seeding. White and black arrowheads indicate air bubbles and deformed layers of coating resin, respectively.

Figure 5

Aggregate formation of various cell types on prevelex-coated microwell array plates and hair follicle neogenesis upon transplantation of aggregates of GFP-labeled cells. (a) Spherical aggregate formation. PS and PDMS plates with square pyramid and hemispherical microwell arrays are coated with prevelex, and the cells indicated are seed and cultured for 1–5 days. (b) Transgenic fetus mice expressing green fluorescence protein (GFP) and hair follicle germ-like aggregates formed at three days of culture. Embryonic epithelial and mesenchymal cells are isolated from the fetus and let them form aggregates in the prevelex-coated PDMS microwell array plate. (c) Lab-made chambers for patch assay with nude mice. (d) Hair follicle neogenesis in vivo. The aggregates composed of the two cell types are transplanted into the dorsal skin of nude mice. Appearance of generated hair shafts is shown. The fluorescent images are from histological sectioning of the transplanted site. Rhodamine-phalloidin and DAPI staining were used to visualize actin cytoskeleton and nuclei of all the cells in the skin sections. GFP indicates cells from grafts.