Generation of a 3D Outer Blood-Retinal Barrier with Advanced Choriocapillaris and Its Application in Diabetic Retinopathy in a Microphysiological System.

The outer blood-retinal barrier (oBRB) provides an optimal environment for the function of the photoreceptor by regulating the exchange of molecules between subretinal space and the choriocapillaris, and its dysfunction could impair the photoreceptor's function and vision. The existing in vitro models have limitations in reproducing the barrier function or physiological characteristics of oBRB and choriocapillaris. Here, we engineered a microphysiological system-based oBRB-choriocapillaris model that simultaneously incorporates the desired physiological characteristics and is simple to fabricate. First, we generated microvascular networks to mimic choriocapillaris and investigated the role of fibroblasts in vasculogenesis. By adding retinal pigment epithelial cells to one side of blood vessels formed with endothelial cells and fibroblasts and optimizing their culture medium conditions, we established an oBRB-choriocapillaris model. To verify the physiological similarity of our oBRB-choriocapillaris model, we identified the polarization and expression of the tight junction of the retinal pigment epithelium, Bruch's membrane, and the fenestral diaphragm of choriocapillaris. Finally, we tried to recapitulate the diabetes mellitus environment in our model with hyperglycemia and diabetes-related cytokines. This induced a decrease in tight junction integrity, loss of barrier function, and shrinkage of blood vessels, similar to the in vivo pathological changes observed in the oBRB and choriocapillaris. The oBRB-choriocapillaris model developed using a microphysiological system is expected to offer a valuable in vitro platform for retinal and choroidal vascular diseases in preclinical applications.

Microfluidic outer blood-retinal barrier model for inducing wet age-related macular degeneration by hypoxic stress.

Wet age-related macular degeneration (AMD) is a severe ophthalmic disease that develops in the outer blood-retinal barrier (oBRB), involving two types of cells, the retinal pigment epithelium (RPE) and the choriocapillaris endothelium (CCE). Unfortunately, the pathogenesis of AMD is unclear, and the risk of the only effective therapy (Anti-VEGF injection) has been consistently argued. Also, since oBRB is hard to observe in vivo, an in vitro model for the pathological study is necessary. Here, we propose an advanced oBRB model, enhanced in two major ways: fully vascularized CCE and the in vivo analogous distance between RPE and CCE. Our model consists of an RPE (ARPE-19) monolayer with adjacent CCE (HUVEC) embedded fibrin gel in the microfluidic chip and required four days to construct an oBRB. Notably, the intercellular distance was tuned to the in vivo scale (<100 µm) without any extraneous scaffold in between. Thus, the two cell layers can interact freely through the extracellular matrix (ECM) in vivo. This is significant as wet AMD is mainly developed through broken intercellular interaction. Thanks to this in vivo similarity, the model incubated under hypoxic conditions, similar to an oxygen-induced retinopathy animal model, showed upregulated vascularization comparable to the AMD condition. We envisage that our model can be used to assist the investigation of AMD.

Microfluidic-based observation of local bacterial density under antimicrobial concentration gradient for rapid antibiotic susceptibility testing.

The need for accurate and efficient antibiotic susceptibility testing (AST) has been emphasized with respect to the emerging antimicrobial resistance of pathogenic bacteria which has increased over the recent decades. In this study, we introduce a microfluidic system that enables rapid formation of the antibiotic concentration gradient with convenient bacterial growth measurement based on color scales. Furthermore, we expanded the developed system to analyze combinatory effects of antibiotics and measured the collective antibiotic susceptibility of bacteria compared to single microfluidic AST methods. By injecting a continuous flow precisely into the channel, the system enabled the concentration gradient to be established between two parallel channels of different antibiotic concentrations within 30 min, before bacteria enter the exponential growth phase. Moreover, the local bacterial growth levels under antibiotic gradient were quantitatively determined by calculating the position-specific grayscale values from the microscopic images and were compared with the conventional optical density measurement method. We tested five antibiotic types on our platform for the pathogenic Gram-negative bacteria strain Pseudomonas aeruginosa, and we were able to determine the minimum inhibitory concentration (MIC) at which 90% to 95% of bacterial growth was inhibited. Finally, we demonstrated the efficacy of our system by showing that most of the antibiotic MICs determined in our platform show good agreement with the MIC range suggested by the Clinical and Laboratory Standards Institutes.