Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

Application of deep UV resonance Raman spectroscopy to column liquid chromatography: Development of a low-flow method for the identification of active pharmaceutical ingredients.

In this study, deep UV resonance Raman spectroscopy (DUV-RRS) was coupled with high performance liquid chromatography (HPLC) to be applied in the field of pharmaceutical analysis. Naproxen, Metformin and Epirubicin were employed as active pharmaceutical ingredients (APIs) covering different areas of the pharmacological spectrum. Raman signals were successfully generated and attributed to the test substances, even in the presence of the dominant solvent bands of the mobile phase. To increase sensitivity, a low-flow method was developed to extend the exposure time of the sample. This approach enabled the use of a deep UV pulse laser with a low average power of 0.5 mW. Compared to previous studies, where energy-intensive argon ion lasers were commonly used, we were able to achieve similar detection limits with our setup. Using affordable lasers with low operating costs may facilitate the transfer of the results of this study into practical applications.

Enhancement of lacrimal gland cell function by decellularized lacrimal gland derived hydrogel.

Sustainable treatment of aqueous deficient dry eye (ADDE) represents an unmet medical need and therefore requires new curative and regenerative approaches based on appropriatein vitromodels. Tissue specific hydrogels retain the individual biochemical composition of the extracellular matrix and thus promote the inherent cell´s physiological function. Hence, we created a decellularized lacrimal gland (LG) hydrogel (dLG-HG) meeting the requirements for a bioink as the basis of a LG model with potential forin vitroADDE studies. Varying hydrolysis durations were compared to obtain dLG-HG with best possible physical and ultrastructural properties while preserving the original biochemical composition. A particular focus was placed on dLG-HG´s impact on viability and functionality of LG associated cell types with relevance for a futurein vitromodel in comparison to the unspecific single component hydrogel collagen type-I (Col) and the common cell culture substrate Matrigel. Proliferation of LG epithelial cells (EpC), LG mesenchymal stem cells, and endothelial cells cultured on dLG-HG was enhanced compared to culture on Matrigel. Most importantly with respect to a functionalin vitromodel, the secretion capacity of EpC cultured on dLG-HG was higher than that of EpC cultured on Col or Matrigel. In addition to these promising cell related properties, a rapid matrix metalloproteinase-dependent biodegradation was observed, which on the one hand suggests a lively cell-matrix interaction, but on the other hand limits the cultivation period. Concluding, dLG-HG possesses decisive properties for the tissue engineering of a LGin vitromodel such as cytocompatibility and promotion of secretion, making it superior to unspecific cell culture substrates. However, deceleration of biodegradation should be addressed in future experiments.

Modulation of Decellularized Lacrimal Gland Hydrogel Biodegradation by Genipin Crosslinking.

Purpose: Hydrogels derived from decellularized tissues are promising biomaterials in tissue engineering, but their rapid biodegradation can hinder in vitro cultivation. This study aimed to retard biodegradation of a hydrogel derived from porcine decellularized lacrimal glands (dLG-HG) by crosslinking with genipin to increase the mechanical stability without affecting the function and viability of lacrimal gland (LG)-associated cells. Methods: The effect of different genipin concentrations on dLG-HG stiffness was measured rheologically. Cell-dependent biodegradation was quantified over 10 days, and the impact on matrix metalloproteinase (MMP) activity was quantified by gelatin and collagen zymography. The viability of LG epithelial cells (EpCs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) cultured on genipin-crosslinked dLG-HG was assessed after 10 days, and EpC secretory activity was analyzed by ß-hexosaminidase assay. Results: The 0.5-mM genipin increased the stiffness of dLG-HG by about 46%, and concentrations > 0.25 mM caused delayed cell-dependent biodegradation and reduced MMP activity. The viability of EpCs, MSCs, and ECs was not affected by genipin concentrations of up to 0.5 mM after 10 days. Moreover, up to 0.5-mM genipin did not negatively affect EpC secretory activity compared to control groups. Conclusions: A concentration of 0.5-mM genipin increased dLG-HG stiffness, and 0.25-mM genipin was sufficient to prevent MMP-dependent degradation. Importantly, concentrations of up to 0.5-mM genipin did not compromise the viability of LG-associated cells or the secretory activity of EpCs. Thus, crosslinking with genipin improves the properties of dLG-HG for use as a substrate in LG tissue engineering.