Neural tissue engineering and three-dimensional in vitro tissue modeling require the development of biomaterials that take into account the specified requirements of human neural cells and tissue. In this study, an alternative method of producing biomimetic hydrogels based on gellan gum (GG) was developed by replacing traditional crosslinking methods with the bioamines spermidine and spermine. These bioamines were proven to function as crosslinkers for GG hydrogel at +37 °C, allowing for the encapsulation of human neurons. We studied the mechanical and rheological properties of the formed hydrogels, which showed biomimicking properties comparable to naïve rabbit brain tissue under physiologically relevant stress and strain. Human pluripotent stem cell-derived neuronal cells demonstrated good cytocompatibility in the GG-based hydrogels. Moreover, functionalization of GG hydrogels with laminin resulted in cell type-specific behavior: neuronal cell maturation and neurite migration.

The future of tissue engineering applications for neuronal cells will require a supportive 3D matrix. This particular matrix should be soft, elastic and supportive for cell growth. In this study, we characterized the suitability of a 3D synthetic hydrogel matrix, PuraMatrix™, as a growth platform for human embryonic stem cell (hESC)-derived neural cells. The viability of the cells grown on top of, inside and under the hydrogel was monitored. The maturation and electrical activity of the neuronal networks inside the hydrogel were further characterized. We showed that cells stayed viable on the top of the PuraMatrix™ surface and growth of the neural cells and neural processes was good. Further, hESC-derived neurons, astrocytes and oligodendrocytes all grew, matured and migrated when cultured inside the hydrogel. Importantly, neuronal cells were able to form electrically active connections that were verified using microelectrode array. Thus, PuraMatrix is a good supportive growth matrix for human neural cells and may serve as a matrix for neuronal scaffolds in neural tissue engineering.

Regenerative medicine, especially cell therapy combined with a supportive biomaterial scaffold, is considered to be a potential treatment for various deficits in humans. Here, we have produced and investigated the detailed properties of injectable hydrazone crosslinked hyaluronanpolyvinyl alcohol (HA-PVA) and alginate-polyvinyl alcohol (AL-PVA) hydrogels to be used as a supportive biomaterial for 3D neural cell cultures. To the best of our knowledge, this is the first time the polymerization and properties of hydrazone crosslinked AL-PVA hydrogel have been reported. The effect of the degree of substitution and molecular weight of the polymer components as well as the polymer concentration of the hydrogel on the swelling, degradation and mechanical properties of the hydrogels is reported. Furthermore, we studied the effect of the above parameters on the growth of human pluripotent stem cell-derived neuronal cells. The most neural cell supportive HA-PVA hydrogel was composed of high molecular weight HA component with brain-mimicking mechanical properties and decreased polymer concentration. AL-PVA hydrogel, with stiffness quite similar to brain tissue, was also shown to be similarly supportive. Neuronal spreading and 3D network formation was enhanced inside the softest hydrogels.

As the complex structure of nervous tissue cannot be mimicked in two-dimensional (2D) cultures, the development of three-dimensional (3D) neuronal cell culture platforms is a topical issue in the field of neuroscience and neural tissue engineering. Computer-assisted laser-based fabrication techniques such as direct laser writing by two-photon polymerization (2PP-DLW) offer a versatile tool to fabricate 3D cell culture platforms with highly ordered geometries in the size scale of natural 3D cell environments. In this study, we present the design and 2PP-DLW fabrication process of a novel 3D neuronal cell culture platform based on tubular microtowers. The platform facilitates efficient long-term 3D culturing of human neuronal cells and supports neurite orientation and 3D network formation. Microtower designs both with or without intraluminal guidance cues and/or openings in the tower wall are designed and successfully fabricated from Ormocomp. Three of the microtower designs are chosen for the final culture platform: a design with openings in the wall and intralumial guidance cues (webs and pillars), a design with openings but without intraluminal structures, and a plain cylinder design. The proposed culture platform offers a promising concept for future 3D cultures in the field of neuroscience.

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell‐, biomaterial‐, and environment‐related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan–poly(vinyl alcohol) (HA1‐PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1‐PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1‐PVA‐col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D.

Stem cell transplantations for spinal cord injury (SCI) have been studied extensively for the past decade in order to replace the damaged tissue with human pluripotent stem cell (hPSC)-derived neural cells. Transplanted cells may, however, benefit from supporting and guiding structures or scaffolds in order to remain viable and integrate into the host tissue. Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. All cell types are shown to efficiently spread over the nanofiber platform and orient according to the fiber alignment. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment. In this work, clinically relevant materials and substrates for nanofibers, fiber coatings, and hydrogel scaffolds are used and combined with cells suitable for developing functional cell grafts for SCI repair.

New strategies for spatially controlled growth of human neurons may provide viable solutions to treat and recover peripheral or spinal cord injuries. While topography cues are known to promote attachment and direct proliferation of many cell types, guided outgrowth of human neurites has been found difficult to achieve so far. Here, three-dimensional (3D) micropatterned carbon nanotube (CNT) templates are used to effectively direct human neurite stem cell growth. By exploiting the mechanical flexibility, electrically conductivity and texture of the 3D CNT micropillars, a perfect environment is created to achieve specific guidance of human neurites, which may lead to enhanced therapeutic effects within the injured spinal cord or peripheral nerves. It is found that the 3D CNT micropillars grant excellent anchoring for adjacent neurites to form seamless neuronal networks that can be grown to any arbitrary shape and size. Apart from clear practical relevance in regenerative medicine, these results using the CNT based templates on Si chips also can pave the road for new types of microelectrode arrays to study cell network electrophysiology.

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