We evaluated the potential of alginate film incorporating ferric ions as a gelling agent (Fe-alginate) in comparison with that incorporating calcium ions (Ca-alginate) as a scaffold for culturing normal human dermal fibroblasts (NHDF). NHDF adhered to Fe-alginate and proliferated well, but no growth of the cells was observed on Ca-alginate. Since vitronectin and fibronectin play pivotal roles in cellular adhesion, their participation in NHDF behavior on alginate surfaces was investigated. We found that vitronectin was a critical element for initial attachment and spreading of NHDF on Fe-alginate. The surface properties of both alginate films were characterized in terms of protein adsorption ability and surface wettability, and it was revealed that Fe-alginate film adsorbed a significantly higher amount of proteins, including vitronectin and fibronectin, and had a higher surface hydrophobicity than Ca-alginate film. Moreover, under serum-free conditions, only a small number of NHDF were able to attach to the surface of Fe-alginate. Fe-alginate appeared to provide an appropriate surface for cellular attachment by adsorption of serum proteins such as vitronectin. These results suggest that Fe-alginate can serve as a scaffold for human fibroblasts and may be useful for tissue engineering research and other biomedical applications.
We investigated the suitability of ferric-ion-cross-linked alginates (Fe-alginate) with various proportions of L-guluronic acid (G) and D-mannuronic acid (M) residues as a culture substrate for human dermal fibroblasts. High-G and high-M Fe-alginate gels showed comparable efficacy in promoting initial cell adhesion and similar protein adsorption capacities, but superior cell proliferation was observed on high-G than on high-M Fe-alginate as culture time progressed. During immersion in culture medium, high-G Fe-alginate showed little change in gel properties in terms of swelling and polymer content, but the properties of high-M Fe-alginate gel were altered due to loss of ion cross-linking. However, the degree of cell proliferation on high-M Fe-alginate gel was improved after it had been stabilized by immersion in culture medium until no further changes occurred. These results suggest that the mode of cross-linkage between ferric ions and alginate differs depending on alginate composition and that the major factor giving rise to differences in cell growth on the two types of Fe-alginate films is gel stability during culture, rather than swelling of the original gel, polymer content, or protein adsorption ability. Our findings may be useful for extending the application of Fe-alginate to diverse biomedical fields.
In this study we investigated differences in the characteristics determining the suitability of five types of ion (Fe(3+), Al(3+), Ca(2+), Ba(2+) and Sr(2+))-cross-linked alginate films as culture substrates for cells. Human dermal fibroblasts were cultured on each alginate film to examine the cell affinity of the alginates. Since cell behavior on the surface of a material is dependent on the proteins adsorbed to it, we investigated the protein adsorption ability and surface features (wettability, morphology and charge) related to the protein adsorption abilities of alginate films. We observed that ferric, aluminum and barium ion-cross-linked alginate films supported better cell growth and adsorbed higher amounts of serum proteins than other types. Surface wettability analysis demonstrated that ferric and aluminum ion-cross-linked alginates had moderate hydrophilic surfaces, while other types showed highly hydrophilic surfaces. The roughness was exhibited only on barium ion-cross-linked alginate surface. Surface charge measurements revealed that alginate films had negatively charged surfaces, and showed little difference among the five types of gel. These results indicate that the critical factors of ionically cross-linked alginate films determining the protein adsorption ability required for their cell compatibility may be surface wettability and morphology.
The present study was conducted to assess the efficiency of a novel cell-harvesting method involving dissolution of the culture substrate composed of Fe-alginate (ferric-ion-cross-linked alginate). Cell harvesting is an essential step for recovery of cultured adherent cells, but conventional methods such as trypsinization or scraping cause considerable damage to the cells. We therefore devised an original method for harvesting cultured cells using Fe-alginate films as a culture substrate and then retrieving the cells by disintegration of the alginate gel. Fe-alginate was easily dissolved under physiological conditions by exchange of cross-linked ions using chelating agents such as citrate. The effects of this cell-harvesting method were investigated in comparison with trypsinization and scraping. Cells harvested by dissolution of the Fe-alginate substrate showed high viability and metabolic activity, high recovery rate on subculture, a low degree of membrane damage with superior expression of cell adhesion molecules (beta1 integrin and N-cadherin), high resistance to oxidative stress, and high viability, metabolic activity and recovery rate after cryopreservation, in contrast with the cells harvested by trypsinization or scraping from tissue-culture polystyrene substrates. These results suggest that our new system involving dissolution of Fe-alginate culture substrate is effective for cell harvesting and may be advantageous for biomedical applications.
We investigated the efficacy of three-dimensional porous ferric-ion-cross-linked alginate (Fe-alginate) gels as cell scaffolds, in comparison with calcium-ion-cross-linked alginate (Ca-alginate) gels. In a previous study, we had demonstrated that twodimensional Fe-alginate film was an efficient material for use as a scaffold, allowing good cell adhesion and proliferation, unlike Caalginate film. In the present study, we fabricated three-dimensional porous Fe-and Ca-alginate gels by freeze-drying and evaluated their effects on cultured cells. The Fe-alginate gels showed higher protein adsorption ability than Ca-alginate gels. Cells formed multicellular spheroids in both types of alginate scaffold, but the number of cultured cells increased with culture time on Fealginate porous gels, whereas those on Ca-alginate gels did not. Moreover, it was revealed that the cells on Fe-alginate scaffolds were still viable inside the multicellular spheroids even after cultivation for 14 days. These results suggest that Fe-alginate provides a superior porous scaffold suitable for three-dimensional culture of cells. Our findings may be useful for extending the application of Fe-alginate to diverse biomedical fields.
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