Abstract
Purpose :
Understanding the pathogenesis of glaucoma and evaluating therapeutic solutions in vitro require a systematic platform capable of mimicking the complex in vivo environment. In the presence of an intact inner blood-retinal barrier(iBRB), transendothelial migration of T cells is restricted. Emerging evidence suggests that this mechanism is compromised in glaucoma, but a conventional in vitro model of two-dimensional cell cultures fails to capture such multi-tissue pathophysiology. Recent progress in microfluidics-based cell culturing or “organ-on-a-chip” has made it possible to reproduce organ-level human physiology and better mimic various disease features found in vivo. This presentation will introduce a "retina-on-a-chip" system consisting of microglia, retinal endothelial cells and T cells to study the cascading effect of T-cell-microglia crosstalk across the iBRB during glaucoma progression. The specific roles of three cytokines (CCL3, CCL4 and IL-6) related to microglia activation will also be elucidated.
Methods :
We construct a multi-compartment microfluidic device, with retinal endothelial cells cultured in one compartment to form a confluent monolayer mimicking the iBRB and cytokines or activated microglia housed in an adjacent interconnecting compartment (n=3 chips per group). We perfuse fluorescently labeled CD 4+ T cells into the retinal endothelial compartment and track infiltration events over 24 hours under a live cell microscope. In the end, we collect supernatant and T cells from both compartments for further chracterization. We also perform immunostaining of microglia and retinal endothelial cells on the chip. To examine iBRB barrier properties due to immunoregulation, we perfuse FITC-dextran to measure the iBRB permeability through quantitative imaging.
Results :
Over 24 hours, we observe significant T cell infiltration under the influence of cytokines, confirming a compromised iBRB and a cascade of immune regulations relevant to glaucoma progression.
Conclusions :
Our microfluidic model elucidates the role of microglia in regulating T cell infiltration in greater detail compared to animal models. Thus, it has great potential to become a low-cost platform technology to study fundamental disease mechanisms and accelerate drug development to benefit glaucoma research.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.