Blended alginate/collagen hydrogels promote neurogenesis and neuronal maturation

Abstract

Brain extracellular matrix (ECM) is complex, heterogeneous and often poorly replicated in traditional 2D cell culture systems. The development of more physiologically relevant 3D cell models capable of emulating the native ECM is of paramount importance for the study of human induced pluripotent stem cell (iPSC)-derived neurons. Due to its structural similarity with hyaluronic acid, a primary component of brain ECM, alginate is a potential biomaterial for 3D cell culture systems. However, a lack of cell adhesion motifs within the chemical structure of alginate has limited its application in neural culture systems. This study presents a simple and accessible method of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation under physiological conditions and tests the hypothesis that such a substrate could influence the behaviour of human neurons in 3D culture. Regulation of the gelation process enabled the penetration of collagen fibrils throughout the hydrogel structure as demonstrated by transmission electron microscopy. Encapsulated human iPSC-derived neurons adhered to the blended hydrogel as evidenced by the increased expression of α1, α2 and β1 integrins. Furthermore, immunofluorescence microscopy revealed that encapsulated neurons formed complex neural networks and matured into branched neurons expressing synaptophysin, a key protein involved in neurotransmission, along the neurites. Mechanical tuning of the hydrogel stiffness by modulation of the alginate ionic crosslinker concentration also influenced neuron-specific gene expression. In conclusion, we have shown that by tuning the physicochemical properties of the alginate/collagen blend it is possible to create different ECM-like microenvironments where complex mechanisms underpinning the growth and development of human neurons can be simulated and systematically investigated.

A Custom Ultra-Low-Cost 3D Bioprinter Supports Cell Growth and Differentiation

“…These cells are very delicate, as previously evaluated from in vitro 2D cultures (Kyrousi et al, 2015). To this regard, we preferred to use a low viscosity bioink (Figure 3B), which will produce less shear stress, while in addition, it will reflect the brain matrix stiffness as several other studies have proposed (Crompton et al, 2007;Moxon et al, 2019;Distler et al, 2020). Following the bioprinting process, cultures were maintained in self-renewal conditions and subsequently were stained using antibodies recognizing Pax6, Sox2, GFAP, and Ki67.…”

Advances in Tissue Engineering and Innovative Fabrication Techniques for 3-D-Structures: Translational Applications in Neurodegenerative Diseases

“…Indeed, Her et al showed that mesenchymal stem cells can differentiate into the neuronal lineage in a substrate that present a Young modulus of 1 kPa, while they transformed into glial cells when this parameter is 10 kPa [ 124 ]. Moreover, a recent work has shown that the physicochemical properties of the alginate/collagen blend could resemble the ECM microenvironment, influencing neuronal-specific gene expression [ 125 ]. In particular, it was shown that oligodendrocyte differentiation and maturation in vitro is enhanced by substrates within the reported range of stiffness of the brain [ 126 ].…”

“…This specific fabrication technique was used, and many phenotypic aspects were investigated, such as the analysis of neurite extension and neural maturation on polyethylene glycol [ 152 ] or 3-D gelatin methacrylate [ 153 ] hydrogels. Moreover, iPSC-derived mature neurons were bioprinted and cultivated on hydrogel scaffolds, and many read-outs were evaluated, such as the expression of integrins, the formation of a complex network, and the expression of synaptophysin along the neurites [ 125 ]. iPSCs can also be differentiated into glial cells, which proved to play a pivotal role in the pathogenesis of neurological disorders.…”

Innovations in 3D Tissue Models of Human Brain Physiology and Diseases

“…[300] Another approach to incorporating cell binding domains has been to mix in collagen fibrils, with this collagen/alginate blended hydrogel used to culture neurons generated from human iPSCs. [301] Other researchers have used defined mixtures of nutrients in agarose and alginate hydrogels to generate 3D multi-layered scaffolds that mimic cortical layers using primary rat cortical neurons in a microfluidic device. [302] Though the vast majority of neuronal tissue engineering has been achieved using rodent-derived neurons, combinatorial studies on matrix composition and architecture have sought to provide a more comprehensive understanding of how cellular microenvironment (ECM and signaling molecules) can influence cell fate in 3D cultures.…”

“…[ 300 ] Another approach to incorporating cell binding domains has been to mix in collagen fibrils, with this collagen/alginate blended hydrogel used to culture neurons generated from human iPSCs. [ 301 ] Other researchers have used defined mixtures of nutrients in agarose and alginate hydrogels to generate 3D multi‐layered scaffolds that mimic cortical layers using primary rat cortical neurons in a microfluidic device. [ 302 ]…”