Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior

• Published in: Experimental eye research 2022-07, Vol.220, p.109102-109102, Article 109102
• Main Authors: Strat, Ana N., Kirschner, Alexander, Yoo, Hannah, Singh, Ayushi, Bagué, Tyler, Li, Haiyan, Herberg, Samuel, Ganapathy, Preethi S.
• Format: Article
• Language: English
• Subjects: Biomechanical Strain; Collagen IV; Extracellular matrix; Fibronectin; GFAP; Glaucoma; Reactive gliosis; Transforming growth factor beta 2
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2022.109102

In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGF β2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGF β2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGF β2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHAs retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with increasing process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGF β2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONH astrocyte-encapsulated hydrogel to investigate astrocyte behavior in response to injury. •Optic nerve head astrocytes (ONHA) retain viability and proliferate in photo-crosslinked biopolymer hydrogels.•The hydrogel system allows for astrocytes to achieve stellate morphology and develop process complexity.•TGFB2 induces astrogliosis in ONHA-hydrogels consistent with 2D models.•This novel 3D biomimetic hydrogel system can be used to further investigate ONHA behavior under glaucomatous insult.