Astrocyte 3D culture and bioprinting using peptide functionalized hyaluronan hydrogels

Abstract

Astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the integrity and function of the blood–brain barrier. Astrocytes are also linked to the pathophysiology of various neurodegenerative diseases. Astrocyte function and organization are tightly regulated by interactions mediated by the extracellular matrix (ECM). Engineered hydrogels can mimic key aspects of the ECM and can allow for systematic studies of ECM-related factors that govern astrocyte behaviour. In this study, we explore the interactions between neuroblastoma (SH-SY5Y) and glioblastoma (U87) cell lines and human fetal primary astrocytes (FPA) with a modular hyaluronan-based hydrogel system. Morphological analysis reveals that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by conjugation of cell-adhesion peptides (cRGD and IKVAV) to the hyaluronan backbone. These effects are retained and pronounced in 3D bioprinted structures. Bioprinted FPA using cRGD functionalized hyaluronan show extensive and defined protrusions and multiple connections between neighboring cells. Possibilities to tailor and optimize astrocyte-compatible ECM-mimicking hydrogels that can be processed by means of additive biofabrication can facilitate the development of advanced tissue and disease models of the central nervous system.

Nanocellulose Reinforced Hyaluronan-Based Bioinks

“…We have previously demonstrated that cell adhesions motifs, such as the fibronectin and laminin derived peptides RGD and IKVAV, with terminal azide groups can be easily conjugated to the HA-BCN backbone, resulting in improved cell−hydrogel interactions. 8 Printability. Shear thinning hydrogels are attractive as bioinks for extrusion-based 3D bioprinting since this facilitates the dispensing procedure while allowing for printing with high shape fidelity.…”

“…The rounded fibroblast morphology is likely a result of the lack of cell-adhesion motifs in the hydrogels. We have previously demonstrated that cell adhesions motifs, such as the fibronectin and laminin derived peptides RGD and IKVAV, with terminal azide groups can be easily conjugated to the HA-BCN backbone, resulting in improved cell–hydrogel interactions …”

“…Engineered ECM inspired hydrogels will always have a less complex composition than native ECM, but their properties can be tailored to promote biologically relevant cellular behavior. 8 The advances in 3D cell culture have promoted the use of hydrogel-based scaffolds in several emerging applications, such as drug screening, 9,10 regenerative medicine, 11,12 and 3D bioprinting. 13 For bioprinting, the hydrogels not only must provide a biologically relevant microenvironment but also exhibit rheological properties that support the printing process.…”

“…Not to mention that they are expensive to produce. , Engineered ECM-mimetic hydrogels are interesting alternatives to biosourced ECM, since they allow full control of the included components and high reproducibility with respect to both chemical and mechanical properties. Engineered ECM inspired hydrogels will always have a less complex composition than native ECM, but their properties can be tailored to promote biologically relevant cellular behavior …”

“…We have recently developed ECM mimetic hydrogels based on cyclooctyne-functionalized HA (HA-BCN) cross-linked through a strain-promoted azide–alkyne cycloaddition (SPAAC) reaction using multiarm azide-functionalized PEG (PEG-Az 8 ). , In addition to allow for bioorthogonal cross-linking of the hydrogels, unreacted BCN groups can be utilized for functionalization of the HA with cell adhesion motifs or other functional groups . This versatile hydrogel system has been demonstrated to support the proliferation of several cell lines such as fibroblasts, hepatocytes, and neural cells and has been utilized as a bioink for 3D bioprinting .…”