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Susanne Mohr, Brandon Coughlin; Newly Designed Culture System to Study Effects of Hyperglycemia on the Cross-Talk between Müller and Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5194.
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© ARVO (1962-2015); The Authors (2016-present)
Considering the increasing numbers of diabetic patients there is a dire need to find new therapeutic strategies to effectively treat or even better cure diabetic retinopathy. However, therapies developed using rodent models rarely translate into successful treatments for patients with diabetic retinopathy. Therefore, the goal of this study was to establish a physiologically relevant flow based culture system using human retinal cells that will allow to identify effects of hyperglycemia on human retinal cell cross-talk and to test potential new therapies.
The cell culture system consisting of a pump and a cartridge fits in a regular incubator (370C/5% CO2). The cartridge consists of a network of hollow fiber artificial capillaries surrounded by an extracellular compartment (ECS). The fibers (0.5μm pores) allow for exchange of proteins between inside of the capillary (LS) and the outside (ECS). Human retinal Müller (hMC) and endothelial cells (HREC) were isolated from donors with no history of diabetes and inflammatory diseases. HREC cells (10ml of 3x106 cells/ml HREC suspension) were injected into the LS. hMCs (3x106 cells) from the same donor were added to the ECS. The cartridges were allowed to settle in for 4 weeks. Formation of cells layers was confirmed using electron microscopy (EM). Effects of hyperglycemia and VEGF on permeability were assessed using trypan blue and expressed as mean area under the curve (AUC) ± SDEV.
Under normal (5mmol/l) glucose condition, HREC formed a monolayer within the capillary fiber as determined by EM. HREC also formed a tight barrier. Following VEGF (2ng/ml) injection into the LS, permeability significantly increased (9.47AUC compared to 2.5AUC). Interestingly, hyperglycemia (25mmol/l) itself had no effect on permeability (2.3±0.3AUC vs 2.46±0.4AUC). For co-culture, attachment of hMCs on outside of the fiber was confirmed by EM. Hyperglycemia caused significant and irreversible barrier break down at 4-6 months (2.13AUC vs 4.87AUC) but not an acute breakdown.
We have established a flow based culture system that seems to mimic physiological relevant cross-talk and microenvironment between human retinal Müller and endothelial cells. This system can be used to identify and test new therapies in order to develop more efficient treatments for diabetic patients.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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