Invertebrate Retinal Progenitors as Regenerative Models in a Microfluidic System

Abstract

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by the misdirected and/or insufficient migration of transplanted cells. RPCs must achieve appropriate spatial and functional positioning in host retina, collectively, to restore vision, whereas movement of clustered cells differs substantially from the single cell migration studied in classical chemotaxis models. Defining how RPCs interact with each other, neighboring cell types and surrounding extracellular matrixes are critical to our understanding of retinogenesis and the development of effective, cell-based approaches to retinal replacement. The current article describes a new bio-engineering approach to investigate the migratory responses of innate collections of RPCs upon extracellular substrates by combining microfluidics with the well-established invertebrate model of Drosophila melanogaster. Experiments utilized microfluidics to investigate how the composition, size, and adhesion of RPC clusters on defined extracellular substrates affected migration to exogenous chemotactic signaling. Results demonstrated that retinal cluster size and composition influenced RPC clustering upon extracellular substrates of concanavalin (Con-A), Laminin (LM), and poly-L-lysine (PLL), and that RPC cluster size greatly altered collective migratory responses to signaling from Fibroblast Growth Factor (FGF), a primary chemotactic agent in Drosophila. These results highlight the significance of examining collective cell-biomaterial interactions on bio-substrates of emerging biomaterials to aid directional migration of transplanted cells. Our approach further introduces the benefits of pairing genetically controlled models with experimentally controlled microenvironments to advance cell replacement therapies.

A Micro-Optic Stalk (μOS) System to Model the Collective Migration of Retinal Neuroblasts

“…Developing eye-brain complexes were extracted from third instar larvae grown from the fly stock described using established methods [47] modified by our group for cell recovery and viability [37,40]. In brief, dissection was completed in a laminar flow hood pre-treated with ultraviolet light (λ = 400 nm) (CellGard ES Energy Saver Class II, Type A2) to maintain sterility using autoclaved materials.…”

“…These dimensions are listed in Table 1, and they were chosen to most accurately represent the reported in vivo dimensions obtained via confocal imaging and dissection of this developmental stage [7,52,53]. The microchannels are spaced 35 μm apart to create multiple OS channels for concurrent RNB study to different extracellular conditions with sufficient volume for 15 brain dissections (0.016 μL) per testing condition [37]. Each microchannel of the µOS array represents one OS segment and has a length (L OS ) of 90 µm, depth of 10 µm, and characteristic width of 37 µm (W OS1 ) on the BL side and 35 µm (W OS2 ) on the EID side.…”

“…Inner surfaces of the µOS were coated with a 15 µg/mL solution of Concanavalin A (ConA) (eBioscience, Carlsbad, CA, USA) for two hours and washed with PBS under sterile conditions. This extracellular substrate was selected based on our previous work comparing RNB adhesion and viability upon a panel of substrates [37] as well as numerous citations in the Drosophila literature validating ConA as an appropriate substrate [39,48]. The coating was then aspirated, and the device washed with PBS and placed in a flow hood to air dry for 1 h. A 150-µL cell suspension was flushed into the system (NE-1000, New Era Pump Systems Inc, Farmingdale, NY, USA) at volume flow rates of Q L < 5 µL/min to minimize shear [41,51] and enable RNB adhesion.…”

“…Microfluidics-based systems have also been developed to examine electrical synapses and neuromodulation in retina, as well as enable the size profiling of retinal stem cells [31][32][33]. However, very few projects have used microfluidics to examine the collective migration within developing visual systems [34,35] central to retinal regeneration therapies, and even fewer works have used invertebrate cells to do so [36][37][38][39]. Ours is the first microfluidic environment designed to represent the developing visual system of Drosophila Melanogaster to anatomical scale and used to examine collective migration therein.…”

“…This device thereby provides an important testing system for studying the mechanisms of Drosophila development to advance emerging strategies for retinal regeneration. Previous work from our group [36,37,[40][41][42] has examined the collective migration of clustered retinal progenitors using microfluidic devices with characteristic lengths much larger than the physiological system examined. In this report, we describe a microfluidic device called the microoptic stalk, or μOS, with dimensions approaching those of the developing eye-brain complex of Drosophila.…”