Purpose: The current Mitomycin C treatment paradigm after glaucoma surgery is indiscriminate and kills local cells/tissues, resulting in many significant side effects. Novel wound modulating adjuvants are necessary to improve the success rates of glaucoma surgical interventions. Is it known that elevated levels of transforming growth factor beta (TGFβ1) are present in the eyes of patients with glaucoma. Additionally, the SMAD 2/3 pathway downstream of TGFβ1 is implicated in human tenon’s capsule fibroblast (HTCFs) conversion to myofibroblast which in turn has been implicated in poor surgical outcomes. Thus, pharmaceutical modulation of the TGFBR-SMAD 2/3 pathway may have the potential to lead to improved trabeculectomy outcomes. Current drug development strategies rely on animal and/or cell culture-based models. Animal models, however, are limited by time, cost and interspecies variability. Two-dimensional monolayer cell cultures do not adequately recreate the in vivo microenvironment. We aim to validate a more physiologically relevant drug testing platform and assess several novel anti-fibrotic agents for their capacity to prevent increased resistance to experimental perfusion through a novel, 3D cell culture system. 
Study Design: To validate the platform, HTCFs were perfused with TGFβ1 to recreate the pathological levels seen in glaucomatous patient aqueous humor and compared to vehicle control simulating healthy aqueous humor. Following validation, the TGFBR-SMAD 2/3 pathway was inhibited using verteporfin to demonstrate attenuation of TGFβ1 mediated effects. Cell and tissue morphology were compared between groups. To demonstrate TGFβ1's clinical effect, system hydrostatic pressure afferent to the perfused cell culture chamber was continuously measured over several days as an analogue for intraocular pressure. 
Methods: The model consists of HTCFs cultured within 3D collagen matrices inside a flow chamber slide. Cell culture media was then perfused through the flow chamber at physiological aqueous turnover rate (2.6 µL/min). Light microscopy and confocal microscopy were used to observe cell morphology and tissue architecture changes in response to TGFβ1 and TGFBR-SMAD 2/3 inhibition. Western blot of cell lysate was done to quantify phenotypic changes in response to TGFβ1 and flow. Finally, afferent system pressure was monitored throughout the experiment and compared. 
Results: Confocal microscopy confirmed the presence of a 3D matrix containing HTCFs embedded into the collagen matrix filled the perfusion chamber. Cells treated with TGFβ1 lead to an increase in system pressure over 4 days compared to vehicle control. Western blot for myofibroblastic phenotype markers were increased in cells treated with TGFβ1. Treatment with TGFBR-SMAD 2/3 inhibitors, verteporfin revealed decreased expression of cytoskeletal proteins consistent with myofibroblast conversion inhibition on confocal microscopy. Pre-chamber increases due to TGFβ1 was also attenuated upon verteporfin treatment. 
Conclusions: As TGFβ1 concentration increases within the chamber, fibroblast to myofibroblast conversion is upregulated affecting the characteristics of the extracellular matrix within the chamber and in turn altering the hydraulic properties of the system. HTCFs treated with TGFβ1 in our model reflect physiology seen in aberrant wound healing after glaucoma surgery. Conversely, verteporfin treatment leads to a decrease in cytoskeletal protein expression, more specifically α-SMA and decreasing pre-chamber pressures. Based on our results, this in vitro 3-D system with perfusion capability is suitable for anti-fibrotic drug screening.

Validation of a bioartificial tenon's capsule tissue model for high throughput novel drug testing

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