RESUMO
In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato , Inflamação/patologia , Neuroglia/patologia , Animais , Células Cultivadas , Ácido Hialurônico/química , Oligodendroglia/patologia , Ratos , Ratos Sprague-DawleyRESUMO
This work describes the development of a robust and repeatable in vitro 3D culture model of glial scarring, which may be used to evaluate the foreign body response to electrodes and other implants in the central nervous system. The model is based on methacrylated hyaluronic acid, a hydrogel that may be photopolymerized to form an insoluble network. Hydrogel scaffolds were formed at four different macromer concentrations (0.50, 0.75, 1.00, and 1.50% (w/v)). As expected, the elastic modulus of the scaffolds increased with increasing macromer weight fraction. Adult rat brain tested under identical conditions had an elastic modulus range that spanned the elastic modulus of both the 0.50 and 0.75% (w/v) hydrogel samples. Gels formed with higher macromer weight fraction had decreased equilibrium swelling ratio and visibly thicker pore walls relative to gels formed with lower macromer weight fractions. Mixed glial cells (microglia and astrocytes) were then encapsulated in the HA scaffolds. Viability of the mixed cultures was most stable at a cell density of 1 × 10(7) cells/mL. Cell viability at the highest macromer weight fraction tested (1.50% (w/v)) was significantly lower than other tested gels (0.50, 0.75 and 1.00% (w/v)). The inflammatory response of microglia and astrocytes to a microelectrode inserted into the scaffold was assessed over a period of 2 weeks and closely represented that reported in vivo. Microglia responded first to the electrode (increased cell density at the electrode, and activated morphology) followed by astrocytes (appeared to line the electrode in a manner similar to glial scarring). All together, these results demonstrate the potential of the 3D in vitro model system to assess glial scarring in a robust and repeatable manner.