Microfluidic cell culture model of the ocular fundus

Abstract

Event Abstract Back to Event Microfluidic cell culture model of the ocular fundus Li Jiun Chen1, Shunichi Tsunajima1, Matsuhiko Nishizawa1, Nobuhiro Nagai2, Toshiaki Abe2 and Hirokazu Kaji1 1 Tohoku University, Bioengineering and Robotics, Japan 2 Tohoku University, Clinical Cell Therapy, United Centers for Advanced Research and Translational Medicine, Japan Introduction: Age-related macular disease (AMD) is a common eye condition and a leading cause of vision loss among people age 50 and above[1]. Simple tasks such as driving or reading become impossible without a functional central vision, which is impaired as the result of damages to the macula. The causes leading to AMD includes, but not limited to, family history, and changes in the cellular microenvironments. Wet form AMD is responsible for 90% of the blindness out of all AMD patients[1]; it is marked by the development of subretinal choroidal neovascularization and degeneration of retinal pigmental epithelial (RPE) cells[2]. Although there are many studies aiming at using microdevices to reproduce organ physiologies, knowledge of on-chip models of the eye is limited. Here, microfabrication technology is used to reconstruct ocular fundus tissues (OF) in vitro and to simulate neovascularization in the microfluidic devices. Materials and Methods: The microfluidic device was made up of 2 layers, on each a channel (width 500 µm; height 150 µm) was embedded. A porous membrane (thickness 6.5 µm, hole diameter 10 µm) was sandwiched between the layers. Microchannels and membranes were made of poly(dimethylsiloxane) (PDMS). Retinal pigment epithelial (RPE) cells were introduced into the upper channel of the device and cultured until they were confluent. Thereafter, human umbilical vein endothelial cells (HUVECs) were loaded into the lower channel. The device was turned upside down to allow cells to adhere onto the lower side of the membrane. RPE cells and HUVEC were dyed respectively with Cell TrackerTM green and red and cell growth was recorded under Real-Time Cultured Cell Monitoring System (Fig. 1,2). Before the aforementioned co-culture model, RPE were cultured alone on the upper channel and the VEGF concentration was collected and measured by ELIZA after replacing DME medium High Glucose (20 mM) with DME medium Low Glucose (3 mM). HUVECs were loaded into the lower channel and cultured alone. The upper channel was perfused with 50 ng/ml of VEGF-A165 and the number of HUVECs migrating to the upper channel was analyzed using the same monitoring system. Results and Discussion: Results show that RPE cells responded to the lowered glucose microenvironment by increasing VEGF secretion (58.8 % compared to the control). In addition, RPE cells exhibited a polar secretion of VEGF which is consistent to the cell physiology. The increased number of HUVEC migrating through the porous membrane in response to VEGF gradient signifies neovascularization. Comparing to the control, there was a 31.3 % increase of cell migration (Fig. 3). Conclusions: The physiology of RPE cells and HUVECs is reproduced in the microdevice. The preliminary results suggest that the alteration of the microenvironment induces responses of RPE cells and the significance of VEGF in neovascularization. In the near future, we anticipate studying responses of the co-culture model to hypoxia to characterize the AMD pathology. Figure 1. Schematic representation of the placement of cells. Figure 2. RPE cells were stained and imaged by fluorescent microscopy. Figure 3. Number of cells migrated through the porous membrane were counted and compared against the control.