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.

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The biopharmaceutical industry faces challenges relating to batch quality control and performance of cell growth media for bioreactors. There is an express need for rapid, sensitive analyses and a non-destructive optical technique to prevent costly losses in productivity. Major issues pertain to cell media preparation, including component stoichiometry, as well as time-, light-, oxidation-and temperature-dependent storage issues. This study reports on cell media analyses using patented A-TEEM spectroscopy, which provide rapid (s-min) analyses of all chromophoric and fluorescent compounds in the UV-VIS range with micro-to sub-microgram/L sensitivity to many aromatic compounds. A-TEEM analysis primarily based on the fluorescence EEMs that are corrected for inner-filter effects (IFE) to eliminate or reduce concentration-dependent spectral distortion. Importantly IFE correction facilitates resolution of low-concentration compounds from components at higher concentrations or intensities in the sample. Additionally, A-TEEM analysis can use 'multi-block' data organization to leverage the resolution capacity of the simultaneously acquired, independent absorbance and EEM data sets. The above factors are important because cell media is complex with many overlapping spectral components with widely varying extinction and or fluorescence properties. A-TEEM data analysis in this study focuses on a machine-learning technique known as Extreme Gradient Boosting (XGB) for purposes of both discrimination (XGBDA) and regression (XGBR). We compare results of XGBDA and XGBDR with controlled data sets to other comparable methods including Partial-Least Squares and Support Vector Machine. Key results include comparisons of cell media from different vendors as well time-and light-dependent effects on media storage. We show that XGB analyses of the A-TEEM data can lead to more effective validation for both discrimination and regression models to facilitate cell media characterization important to quality control including the effects of photo-oxidation.