Abstract
Purpose :
It has been suggested that optic nerve head (ONH) astrocytes (ACs) transition to a reactive phenotype when subjected to abnormal deformations, e.g. due to elevated IOP in glaucoma. Reactive ACs may play a role in retinal ganglion cell (RGC) damage. We describe development and characterization of a high-throughput, physiologically relevant, 3D culture system for interrogation of AC mechanobiology as a step towards an “ONH on a chip”.
Methods :
We fabricated a custom bioreactor compatible with a standard 96-well tissue culture plate, which applied cyclic unconfined compression to primary rat ONH ACs cultured in collagen/alginate 3D gel matrices with mechanical properties “tuned” by varying alginate concentration. 3D gel and rat ONH mechanical properties were measured by cyclic compressive testing (CellScale MicroSquisher). The phenotype of primary cells was verified by immunohistochemistry and western blot in both monolayer and 3D cultures. Total protein was extracted from 3D cultures using a Bead Mill Homogenizer. Oxygenation of 3D cultures was assessed using dispersible microsensors and phase fluorimetry (Lesher-Perez 2017), which is not affected by differences in light paths like intensity-based measurements.
Results :
Isolated primary ONH cells in 2D culture were positive for astrocyte markers: glial fibrillary acidic protein (GFAP), vimentin, and connexin 43. Stress-strain curves from cyclic mechanical tests were repeatable by cycle 5, and were fit with a Neo-Hookean material model (Fig 1). Effective stiffness of low alginate gels was comparable to rat ONH (p = 0.50); high alginate gels were 6x stiffer. Bioreactor design and PDMS-based microsensor encapsulation (Fig 2) enabled phase shift measurements of oxygen concentration inside the gels throughout experiments
Conclusions :
We have developed a high throughput platform to study ONH AC mechanobiology, where primary rat ONH ACs are cultured in 3D gels with either physiological or elevated stiffness, and gels are monitored for hypoxia. Previous work demonstrated reduced baseline DiTNC1 cell activation in 3D cultures as compared to 2D. This platform will be used to investigate the role of 3D matrix stiffness in the response of primary ACs to mechanical loading. We hypothesize that higher matrix stiffness will induce higher baseline activation, but reduce strain sensitivity.
Funding: Georgia Research Alliance
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.