The potential of carbohydrate supramolecular hydrogels for long-term 3D culture of primary fibroblasts

N-Alkyl-galactonamides, which are small synthetic molecules derived from galactose, self-assemble to give fibrous hydrogels. These molecules are biocompatible and, in a previous study, the cell culture of human neural stem cells was performed for 7 days on a gel of N-heptyl-d-galactonamide. With the objective of broadening the scope of these molecules as scaffolds for cell culture, in the present study, the culture of primary human dermal fibroblasts has been carried out on N-nonyl-d-galactonamide hydrogels. These supramolecular fibrillar hydrogels have a sufficient mechanical strength to withstand cell culture (approximate to 50 kPa) and they are resistant enough on the long term to carry out the cell culture over at least 3 weeks. In contrast to N-heptyl-d-galactonamide, N-nonyl-d-galactonamide is insoluble in the culture medium. It avoids its dissolution at each renewal of the culture medium. The molecule is only slowly eliminated by other mechanisms (1/3rd in 3 weeks), which did not impair the cell culture on a monthly scale. The hydrogel's microstructure and how the cells organize on this scaffold have been studied using electron and two-photon microscopies. The gel is made of a quite homogeneous network with a width of approximate to 180 nm and hundreds of micrometer long fibers, except at the surface where a dense mat of heterogeneous fibers is formed. We focused on methods able to colocalize the cells and the gel fibers. Many cell clusters have elongated and multidirectionnal shapes, guided by the fibers. Chains of single cells are also found following the fibers from one cluster to another. N-Nonyl-d-galactonamide fibers, which have the advantage of not being autofluorescent, do not mask the fluorescence of cells. But interestingly, they give a strong second harmonic generation (SHG) signal, due to their well-organized lamellar structure. We also made a special effort to visualize the penetration of cells within the depth of the hydrogels, in 3D, notably by sectioning the hydrogels, despite their softness. It was found that most of the cells stayed at the surface, but several cells grew within the supramolecular fiber network between 50 and 100 mu m depth.