Käpylä Elli, Sorkio Anni, Teymouri Shokoufeh, Lahtonen Kimmo, Vuori Leena, Valden Mika, Skottman Heli, Kellomäki Minna, Juuti-Uusitalo Kati
BioMediTech, Tampere, Finland.
Langmuir. 2014 Dec 9;30(48):14555-65. doi: 10.1021/la5023642. Epub 2014 Nov 24.
In in vitro 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, rigid surfaces such as cover glasses. In this study, we assessed how the silanization of glass with 3-aminopropyltriethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (MAPTMS), or polymer-ceramic material Ormocomp affects the surface properties, protein binding, and maturation of hESC-RPE cells. The surface properties were studied by contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a protein binding assay. The cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV-coated untreated or silanized surfaces for 42 days. The Ormocomp treatment significantly increased the hydrophobicity and roughness of glass surfaces compared to the APTES and MAPTMS treatments. The XPS results indicated that the Ormocomp treatment changes the chemical composition of the glass surface by increasing the carbon content and the number of C-O/═O bonds. The protein-binding test confirmed that the Ormocomp-treated surfaces bound more collagen IV than did APTES- or MAPTMS-treated surfaces. All of the silane treatments increased the number of cells: after 42 days of culture, Ormocomp had 0.38, APTES had 0.16, MAPTMS had 0.19, and untreated glass had only 0.062, all presented as million cells cm(-2). There were no differences in cell numbers compared to smoother to rougher Ormocomp surfaces, suggesting that the surface chemistry and, more specifically, the collagen binding in combination with Ormocomp are beneficial to hESC-RPE cell culture. This study clearly demonstrates that Ormocomp treatment combined with collagen coating significantly increases hESC-RPE cell attachment compared to commonly used silanizing agents APTES and MAPTMS. Ormocomp silanization could thus enable the use of microscopic live cell imaging methods for hESC-RPE cells.
在体外活细胞成像中，在诸如盖玻片等薄的、透明的、刚性表面上培养和评估人胚胎干细胞衍生的视网膜色素上皮（hESC-RPE）细胞将是有益的。在本研究中，我们评估了用3-氨丙基三乙氧基硅烷（APTES）、甲基丙烯酸3-(三甲氧基硅基)丙酯（MAPTMS）或聚合物-陶瓷材料Ormocomp对玻璃进行硅烷化处理如何影响hESC-RPE细胞的表面性质、蛋白质结合和成熟。通过接触角测量、X射线光电子能谱（XPS）、原子力显微镜（AFM）和蛋白质结合测定来研究表面性质。通过在未处理或硅烷化的IV型胶原包被表面上培养hESC-RPE细胞42天来评估细胞粘附和增殖。与APTES和MAPTMS处理相比，Ormocomp处理显著增加了玻璃表面的疏水性和粗糙度。XPS结果表明，Ormocomp处理通过增加碳含量和C-O/═O键的数量改变了玻璃表面的化学成分。蛋白质结合试验证实，与APTES或MAPTMS处理的表面相比，Ormocomp处理的表面结合了更多的IV型胶原。所有硅烷处理均增加了细胞数量：培养42天后，Ormocomp处理的表面有0.38（单位：百万个细胞/cm²），APTES处理的表面有0.16，MAPTMS处理的表面有0.19，未处理的玻璃表面仅有0.062。与Ormocomp表面从较光滑到较粗糙相比，细胞数量没有差异，这表明表面化学性质，更具体地说，与Ormocomp结合的胶原结合对hESC-RPE细胞培养有益。本研究清楚地表明，与常用的硅烷化剂APTES和MAPTMS相比，Ormocomp处理与胶原包被相结合显著增加了hESC-RPE细胞的附着。因此，Ormocomp硅烷化可以使hESC-RPE细胞能够使用显微活细胞成像方法。
