Elevated hydrostatic pressure (EHP) systems are the gold standard for modeling ocular hypertension in vitro and provide insight into the molecular mechanisms of glaucoma. We developed a glaucoma-on-a-chip (GOC) system to evaluate the viability of retinal ganglion cells (RGCs) against high pressure as a noxious condition and the potential effect of neuroprotective treatments.

A three-layered chip consisting of interconnecting microchannels and culture wells was designed based on simulation of physical parameters which had been determined during experimental tests. The chip layers were fabricated from poly-methyl methacrylate (PMMA) sheets. Multiple inlet ports allow culture media and gas into the wells under elevated hydrostatic pressure. The bottom surface of the wells was modified by air plasma jet and coated with different membranes to model an extracellular microenvironment. The SH-SY5Y neuroblastoma cell line was initially employed to determine the best supporting membrane. Thereafter, the study experiments were performed using RGCs obtained from postnatal 5-7 Wistar rats purified by magnetic assisted cell sorting (MACS). These cultured cells were exposed to normal (15 mmHg) or elevated pressure (33 mmHg) for 6, 12, 24, 36 and 48 hours, with and without adding brain-derived neurotrophic factor (BDNF).

A membrane formed by PDL/laminin provided the best microenvironment for neuroblastoma cells which served as model cells. Flow cytometry and immunocytochemistry assays demonstrated 70% purification gain for RGCs isolated from Wistar rats by MACS. The isolated RGCs were used for cell viability experiments. RGC survival rates were 85, 78, 70, 67 and 61 percent under normal pressure versus 40, 22, 18, 12 and 10 percent under high pressure at 6, 12, 24, 36 and 48 hours, respectively (P <0.0001). BDNF treatment induced an approximate two-fold decrease in the rate of RGC death under both normal and elevated pressures (p <0.01 to 0.0001).

This GOC model recapitulated the effects of elevated pressure during relatively short time periods and demonstrated the neuroprotective effect of BDNF. Our system may be utilized to implement pre-defined experimental conditions and evaluate specific variables contributing to RGC survival.