Silane-modified substratum improves cell attachment of human embryonic stem cell-derived retinal pigment epithelial cells

Abstract

Purpose: In in vivo assessments, such as live cell imaging, it would be beneficial to grow and assess human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells on thin, transparent and rigid surfaces such as cover glasses. Thus, the objective of this study was to assess how silanization with 3-aminopropyltriethoxysilane (APTES) and 3-(trimethoxysilyl)propyl methacrylate (MAPTMS) or Ormocomp® affects the surface properties of glass, protein binding and finally the maturation of hESC-RPE cells.

Methods: Borosilicate glass coverslips were silanized either with commercially available silanizing agents, APTES or MAPTMS, or Ormocomp®. The properties of the surface treated coverslips were assessed with contact angle measurements, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Lastly, the cell adherence and proliferation were evaluated by culturing hESC-RPE cells on collagen IV coated untreated or silane-treated surfaces for 42 days.

Results: In the contact angle measurements, there was no difference between the untreated, APTES or MAPTMS treated surfaces, but the Ormocomp®-treatment induced a statistically significant increase in the hydrophobicity. XPS results indicated that the Ormocomp® treatment increased the amount of C-O and C=O bonds on the glass surface. All the silane treatments increased surface roughness detected in AFM, although only with Ormocomp® the change was statistically significant. The protein binding test confirmed that the Ormocomp® treated surfaces bound more collagen IV than APTES or MAPTMS treated surfaces. Finally, all the silane treatments increased the number of attached cells when compared to unsilanized substrata. The highest cell numbers were detected on Ormocomp® treated surfaces.

Conclusions: This study clearly demonstrated that Ormocomp® treatment increased the surface hydrophobicity, surface roughness and collagen IV binding compared to the commonly used commercial silanizing agents APTES and MAPTMS. Furthermore, when comparing to the unsilanized or APTES or MAPTMS treated substrata, the Ormocomp® treatment had the most significant effect in augmenting hESC-RPE cell attachment. We can conclude that the Ormocomp® silanization promotes the attachment of hESC-RPE cells on glass, thus enabling the use of microscopic live cell imaging methods also for hESC-RPE cells.

Keywords: 721 stem cells • 701 retinal pigment epithelium • 688 retina