Simple Synthetic Molecular Hydrogels from Self-Assembling Alkylgalactonamides as Scaffold for 3D Neuronal Cell Growth

Abstract

In this work, we demonstrated that the hydrogel obtained from a very simple and single synthetic molecule, N-heptyl-galactonamide was a suitable scaffold for the growth of neuronal cells in 3D. We evidenced by confocal microscopy the presence of the cells into the gel up to a depth of around 200 μm, demonstrating that the latter was permissive to cell growth and enabled a true 3D colonization and organization. It also supported successfully the differentiation of adult human neuronal stem cells (hNSCs) into both glial and neuronal cells and the development of a really dense neurofilament network. So the gel appears to be a good candidate for neural tissue regeneration. In contrast with other molecular gels described for cell culture, the molecule can be obtained at the gram scale by a one-step reaction. The resulting gel is very soft, a quality in accordance with the aim of growing neuronal cells, that requires low modulus substrates similar to the brain. But because of its fragility, specific procedures had to be implemented for its preparation and for cell labeling and confocal microscopy observations. Notably, the implementation of a controlled slow cooling of the gel solution was needed to get a very soft but nevertheless cohesive gel. In these conditions, very wide straight and long micrometric fibers were formed, held together by a second network of flexible narrower nanometric fibers. The two kinds of fibers guided the neurite and glial cell growth in a different way. We also underlined the importance of a tiny difference in the molecular structure on the gel performances: parent molecules, differing by a one-carbon increment in the alkyl chain length, N-hexyl-galactonamide and N-octyl-galactonamide, were not as good as N-heptyl-galactonamide. Their differences were analyzed in terms of gel fibers morphology, mechanical properties, solubility, chain parity, and cell growth.

3D printing of a biocompatible low molecular weight supramolecular hydrogel by dimethylsulfoxide water solvent exchange

“…Solutions of N-heptyl-ᴅ-galactonamide in dimethylsulfoxide are prepared by dissolving the powder at 2.5 wt% (25 mg/mL) at room temperature with the help of an ultrasound bath. The synthesis of N-heptyl-ᴅ-galactonamide has been described elsewhere 40 or it can be purchased from Innov'Orga, Reims, France. Dimethylsulfoxide has been purchased from Fisher, quality 99 %, non-anhydrous.…”

“…In our preceding work, we showed that low molecular weight hydrogels of N-alkylglyconamides and more especially, N-heptyl-D-galactonamide (GalC7), are biocompatible and allow the growth and the differentiation of human neural stem cells into neurons and glial cells 40 . For different applications, we were looking for methods that would enable to inject or extrude those gels.…”

Wet spinning and radial self-assembly of a carbohydrate low molecular weight gelator into well organized hydrogel filaments

“…7g). 2 Some of these fibers reach a millimetric length and several microns of width. The sol-gel transition occurs at high temperature, around 65°C, precluding notably the introduction of living matter before setting.…”

“…Yet it has been shown that the structure of these fibers at the micrometric/nanometric scale has an impact on the behavior of the cells seeded in these gels. 1,2 Several LMWGs are suitable for cell culture. [3][4][5][6][7][8] Most of them are based on peptides, but other families of LMWGs are also promising for these applications, based on carbohydrates, 2,9-16 nucleosides [17][18][19][20][21][22][23] or other building blocks.…”

“…293)which is easy to produce at the gram scaleprovided good results for the culture of a neuronal cell line (Neuro2A) and adult human neural stem cells (hNSCs). 2 It has been related notably to the very low rigidity of these gels, which is known to be suitable for the growth and differentiation of neuronal cells. 25 Still in the context of neuronal cell culture, it has been shown that oriented scaffolds are able to guide the development of neurites and axons in one preferential direction and can help in a suitable tissue reconstruction.…”