Retinal synaptic regeneration via microfluidic guiding channels

Retinal degeneration is a leading cause of blindness that, together with glaucoma, retinitis pigmentosa, and age-related macular degeneration, will affect 196 million people worldwide in 20201. The thickness of human retina approximately varies from 100 micron at periphery and 250 micron at optic nerve head and the retina is composed of three layers (from the interior to the exterior surface: the ganglion neurons, bipolar neurons, and photoreceptors). A large number of degenerative retinal diseases are associated with the degeneration of these three layers2. To date, there have been few therapeutic options for patients with retinal degeneration. One potential strategy for treatment of this condition is cell transplantation to regenerate retinal tissue. Recent studies have demonstrated the potential of replacing degenerated photoreceptors by injection of either immature rod photoreceptors or engineered stem cells3,4,5. Successful cell therapy based on transplantation of retinal precursors derived from postnatal retinal cells or embryonic stem cells is critically dependent on migration, integration, and maturation of the transplanted cells6,7. In addition, it has been shown in various mouse eye disease models that the developmental stage of transplanted retinal precursors is important to the success of integration and maturation of donor and host cells8,9. However, it remains challenge to increase the efficiency of integration and maturation in mouse models as well as in human clinical trials. To date, only a limited number of patients have experienced improved eyesight following transplantation of retinal precursor cells (derived from human embryonic stem cells) into degenerated retinal tissue5,10. It is still largely unknown how donor and host cells communicate with each other. Therefore, there is a critical need to develop an in vitro platform that can mimic retinal function, permit investigation of communication between transplanted and host cells, and facilitate high-throughput screening of retinal regenerative factors at the cellular level to improve the success rate of cell therapy. Communication between the layered retinal neurons consists of sending and receiving signals through synapses, which are specialized intercellular junctions11. In vitro reconstruction of synaptic connections is an important approach to investigate retinal neuron communication. Traditionally, the study of retinal intercellular communications has been conducted in petri dishes with a random distribution of cells. However, it is difficult to mimic the layered retinal structure, delineate the topologic effect, control the direction of synapses, and quantify changes in synaptic connectivity. Recently, micro-fabricated channels have been used to guide neuronal growth and generate an organized network of synaptic connections12,13,14, particularly for brain neurons. However, to the best of our knowledge, no study has reported synaptic regeneration of retinal neurons using microfabricated channels with geometric guidance. Therefore, we hypothesize that the designed microchannels might facilitate regeneration of the retinal synaptic connections and reconstruction of the in vivo functions. Here, we present a new method for quantitative analysis of retinal synaptic regeneration (RSR) through image-based counting of connected microchannels in a microfluidic chip (RSR-Chip). In this study, multiple arrays of microchannels with the dimensions 50 or 100 μm long, 3 or 4 μm wide, and 17 μm high were utilized to reconstruct the retinal neuronal synapse and form an organized network using R28 retinal precursor cells. R28 cells are derived from postnatal mouse retina and are a popular model for investigation of retinal cell therapy because they differentiate into photoreceptors, Muller cells, and ganglion cells3,9,15. The length of the microchannels can recapitulate the short-range (50–100 μm) interactions of retinal cells, such as amacrine-bipolar cell interactions16. We demonstrate that the synaptic connections of the precursor cells regenerated and formed a network of oriented synapses in the RSR-Chip. The functions of the regenerated synapses were confirmed by immunodetection of phosphorylated extracellular signal-regulated kinase (pERK) of retinal precursors. Furthermore, the effects of inhibitory and excitatory molecules on the dynamics of synaptic regeneration were assessed.