人類黑色素細胞於高分子生醫材料上之研究

Abstract

This assay was aimed to develop a cellular patch with melanocyte spheroids so as to improving the drawbacks encountered in autologous melanocyte transplantation for vitiligo treatment by using melanocyte suspension. The mechanism underlying melanocyte spheroid formation was unclear, and the cellular patch was not evaluated in vitro before clinical application. Therefore, several materials and different topological structures were chosen and designed for culturing melanocytes with kinds of morphologies, spheroids, and monolayers. The findings demonstrated that the topological structure and spheroid-forming factor are not pertinent for melanocyte spheroid formation in two-dimensional cultures. Detail analysis showed that melanocytes displayed in form of different morphologies resulting from cell–substrate interaction; single cell displayed rough, bipolar, or dendritic morphologies, and multiple cells showed monolayers, attached spheroids, or suspended spheroids. Furthermore, this study disclosed that upside-down method is feasible to evaluate cell–substrate interactions between two different substrates, which corresponds with the relative strengths of cell–cell and cell–substrate interactions. A new model was successfully established to evaluate the upside-down cellular patch in vitro before clinical application. Though reported limitedly in tissue engineering, modification of cellular functions can be achieved by culturing them into multicellular spheroids. We have shown melanocytes form spheroids on chitosan surface. However, how biomaterials promote spheroid formation has never been systemically investigated. In this work, nylon, which inhibits melanocyte spheroid formation, and chitosan, which promotes melanocyte spheroid formation, are used to prepare nylon/chitosan-blended membranes. Membranes composed of pure nylon, pure chitosan and various ratios of nylon and chitosan are employed to examine their effects on spheroid formation. Melanocytes show better adhesion to nylon membranes than that to chitosan membranes. In blended membranes, as more nylon is incorporated, cell adhesion increases and the trend for spheroid formation decreases. Melanocytes can only form spheroids on membranes with poorer cell adhesion. Examining the surface of the blended membranes shows phase separation of nylon and chitosan. As nylon content increases, the nylon phase on the membrane surface increases and thereby enhances cell adhesion. The opposite trend for cell adhesion and spheroid formation substantiates our hypothesis of spheroid formation on biomaterials: a balance between cell–substrate interaction and cell–cell interaction. The decrease in cell–substrate interaction tilts the balance to a state more favorable for spheroid formation. Our work can serve as a model to investigate the relative strengths of cell–cell and cell–substrate interactions and pave way to design blended membranes with desired physical properties while preserving the spheroid-forming activity. Cell behaviors in three-dimensional spheroids are known to be different from those in monolayer cultures; however, very few studies have compared the characteristics of cell spheroids formed through different biomaterial-induced processes. This study investigated the mechanism of melanocyte spheroid formation by using membranes composed of two hydrophilic polymer-based biomaterials, namely chitosan and polyvinyl alcohol (PVA). Our findings revealed that different spheroid-forming processes occurred on the two biomaterials. Human melanocytes were provided by the cell bank of the Department of Dermatology, National Taiwan University Hospital. The cell viability was determined through the MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide; Sigma) colorimetric assay. The cell living rate was determined using the trypan blue exclusion test. The amount of fibronectin adsorbed was quantified through Western blot analysis. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Duncan’s test, and P values <0.05 or <0.01 was considered significant. In the study, the melanocytes attached to, migrated on, and aggregated on the chitosan surface and then formed spheroids. By contrast, on the PVA surface, the melanocytes directly aggregated to form three-dimensional spheres in suspension. The proliferative ability and survival rate of the melanocytes were considerably higher on the chitosan membranes than on the PVA membranes. We concluded that only cell–cell interactions dominated in melanocytes seeded on the PVA membrane, whereas cell–cell and cell–substrate interactions occurred on the chitosan membranes and further enhanced cellular functions. The chitosan-induced spheroids could probably overcome the diffusion and assimilation of trophic factors.