Retina-on-a-chip: a microfluidic platform for point access signaling studies

Abstract

We report on a microfluidic platform for culture of whole organs or tissue slices with the capability of point access reagent delivery to probe the transport of signaling events. Whole mice retina were maintained for multiple days with negative pressure applied to tightly but gently bind the bottom of the retina to a thin poly-(dimethylsiloxane) membrane, through which twelve 100 μm diameter through-holes served as fluidic access points. Staining with toluidine blue, transport of locally applied cholera toxin beta, and transient response to lipopolysaccharide in the retina demonstrated the capability of the microfluidic platform. The point access fluidic delivery capability could enable new assays in the study of various kinds of excised tissues, including retina.

Keywords: Microenvironment; Microfluidic tissue culture; Retina.

Fig. 1

Retina-on-a-chip microfluidic platform is shown in (a) exploded view with media cylinder broken into the retina well, agar gel (green, at the top), and glass cylinder, (b) top view with a PDMS disk substituting for the retina and media cylinder removed to show the path of fluid flow within the device, and (c) assembled view with media in channels. d Close-up schematic of the layout of access channels and suction channel in the thin film PDMS layer. All channels are 300 μm wide and all through-holes are 100 μm in diameter. e Schematics of deflection of retina into through-holes in PDMS thin film layer. Without flow or suction, the retina does not deform and seal the through-hole and channel. When negative pressure is applied to the outlet tubing via a syringe, the retina deforms into the through-hole sealing the access channel and allowing for fluid flow. Note that the retina deforms in both the access and suction channels to seal the through-holes due to flow through the channel or suction pressure in the channel, respectively

Fig. 2

Once the retina is placed onto the device, the nerve head can be found with an array of surrounding vessels, vasculature, and RGC axons. The visibility of such anatomy within the retina shows there was no gross deformation of the major anatomical structures after explant on the device. a Image taken at the focal plane of the retina to show the detailed retina components. b Image taken at a focal plane closer to the thin film PDMS layer to demonstrate placement on the device. (Inset) Retina stained with toluidine blue. Note that the vessels and RGC axons do not take up the dye, but the surrounding cells are stained. Scale bar =100 μm

Fig. 3

Demonstration of suction pressure and fluid flow through the device. a Retina is at rest with no suction applied to the channels. b Negative pressure is gently applied in the suction channel. c When the retina deforms into the through-holes in the suction channel, GFP+ glial cells are seen in the focal plane of the thin film PDMS layer. d Toluidine blue is applied to one of the access channels by pulling fluid through the device via a needle and syringe attached by tubing. Note there is no leakage from the through-hole due to the retina deformation creating a seal. e The toluidine blue in the access channel is replaced by media leaving a small and focused stain on the retina. f If the device is not used properly and negative pressure is not used to create a seal between the tissue and through-hole, dye will leak throughout the device staining a much larger area than needed. Scale bar =100 μm

Fig. 4

a Cholera toxin beta subunit (CTB), a protein related to cholera infection, was added to one of the access channels and the retina was cultured for 24 h. The CTB in the channel was then flushed with culture media and imaged. b Note some excess CTB remains in the channel in the vicinity of the through-hole after flushing. c Close up image of the RGC staining at the through-hole in (a). RGC somas (bold arrow) that were stained demonstrate axonal transport of the CTB

Fig. 5

a Red CTB was added to the media cylinder to stain the entire retina while green CTB was added to access channels 4 days after explanting to demonstrate successful culture on the microfluidic platform. After culture for 24 h, the green CTB in the channels was replaced with media. Note that CTB can only be absorbed by living cells, thus the retina was shown to be healthy after 4 days in culture. (b–d) Shown in larger images are the three through-holes where green CTB has stained the healthy retinal ganglion cells in the retinal area within and immediately surrounding the access point. e The mean distance of green CTB diffusion and labeling from edge of access point is restricted to an average of 40 μm over 16 h

Fig. 6

a–c GFP+ microglia in retina from CX3CR1-GFP mice with exposure to culture media only are imaged over 30 min. Note the microglia in the circle, which indicates the through-hole, do not migrate directionally throughout the 30 min of imaging. ONH denotes the optical nerve head of the retina tissue. d GFP+ microglia surrounding an access point (Region 1) in retina from CX3CR1-GFP mice prior to LPS delivery. e Activation and migration of microglia to access point at 1 min and f 30 min post-LPS delivery to access point. Solid lines delineate edge of microfluidic channel. Dotted lines indicate the boundaries of the four sampling regions. g Five hand-counted sample sets were taken at each time point and averaged to give the number of fluorescently labeled microglia in each region. Evidence of immediate microglia migration was observed at 1 min in regions closest to LPS application and delayed migration toward the point of application was observed in regions farther from LPS exposure at 30 min. The total number of microglia at each time point is displayed in the legend. Scale bar (a–c) = 200 μm. Scale bar (d–f) = 50 μm