Abstract
Elucidation of the basic mechanisms underlying human disease pathogenesis depends on the findings afforded to us through in vivo and in vitro approaches. While there are inherent limitations in any model system, 2D in vitro culture systems tend to be particularly restricted due to their static nature. Here, we adapted a flow-based hollow-fiber cartridge system to better understand the cellular influences of human retinal microvascular endothelial cells and mouse-derived neutrophils under high glucose conditions similar to those observed in diabetes. Analyses by western blot and flow cytometry indicate that pro-inflammatory molecules known to be associated with the pathogenesis of diabetic retinopathy were significantly elevated following high glucose exposure, including VEGF, ICAM-1, and ROS. Changes in mitochondrial potential were also observed. Further, we demonstrate that this innovative system allows for cross-species co-culture as well as long-term culturing conditions. This in vitro modeling system not only mimics the retinal microvasculature, it also allows for the examination of cellular interactions and mechanisms that contribute to diabetic retinopathy, a visually debilitating complication of diabetes.
Highlights
Microvascular pathology is well associated with the development and progression of DR
The current study uses the flow-based hollow-fiber modeling system, which is modeled after the mammalian circulatory system and mimics retinal vasculature conditions in vivo to better understand how this cellular interaction and high glucose conditions contribute to DR pathogenesis
Using the flow-based hollow-fiber system, we first cultured human retinal endothelial cells (HRECs) alone to establish that these cells are responding to normal and high glucose conditions to that which is observed under static culture conditions and in in vivo murine models of streptozotocin (STZ)-induced DR
Summary
Microvascular pathology is well associated with the development and progression of DR. Stages of pathology include (but are not limited to): acellular capillary formation, cell death (both retinal endothelial cells [RECs] and associated pericytes), subclinical inflammation and leukostasis[9]. Given the close interaction between RECs and infiltrating inflammatory cells in the retinal vasculature during the progression of DR, it is imperative to generate in vitro conditions that closely mimic what is observed in vivo in order to appropriately examine this interrelationship. In vitro analysis is largely limited to traditional 2D static culture conditions, which poorly mimic the dynamic environment within the human body. The current study uses the flow-based hollow-fiber modeling system, which is modeled after the mammalian circulatory system and mimics retinal vasculature conditions in vivo to better understand how this cellular interaction and high glucose conditions contribute to DR pathogenesis. Hollow fiber bioreactors are not a new technique per se, this is the first study to establish a co-culture system between HREC and mouse-derived PMN under such a physiologically dynamic environment
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