The devastating effects and incurable nature of hereditary and sporadic retinal diseases such as Stargardt disease, age-related macular degeneration or retinitis pigmentosa urgently require the development of new therapeutic strategies. Additionally, a high prevalence of retinal toxicities is becoming more and more an issue of novel targeted therapeutic agents. Ophthalmologic drug development, to date, largely relies on animal models, which often do not provide results that are translatable to human patients. Hence, the establishment of sophisticated human tissue-based in vitro models is of upmost importance. The discovery of self-forming retinal organoids (ROs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) is a promising approach to model the complex stratified retinal tissue. Yet, ROs lack vascularization and cannot recapitulate the important physiological interactions of matured photoreceptors and the retinal pigment epithelium (RPE). In this study, we present the retina-on-a-chip (RoC), a novel microphysiological model of the human retina integrating more than seven different essential retinal cell types derived from hiPSCs. It provides vasculature-like perfusion and enables, for the first time, the recapitulation of the interaction of mature photoreceptor segments with RPE in vitro. We show that this interaction enhances the formation of outer segment-like structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics. In addition, we demonstrate the applicability of the RoC for drug testing, by reproducing the retinopathic side-effects of the anti-malaria drug chloroquine and the antibiotic gentamicin. The developed hiPSC-based RoC has the potential to promote drug development and provide new insights into the underlying pathology of retinal diseases.

The devastating effects and incurable nature of hereditary and sporadic retinal diseases such as Stargardt disease, age-related macular degeneration or retinitis pigmentosa urgently require the development of new therapeutic strategies. Additionally, the prevalence of retinal toxicities is becoming more and more an issue of novel targeted therapeutic agents. To date, (ophthalmologic) drug development largely relies on animal models. Inadequate translatability of results from animal models to humans, however, limits advances in drug development and discovery. Hence, the establishment of more relevant human tissue-based in vitro models is of upmost importance. The discovery of self-forming retinal organoids (ROs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) is a promising approach to model the complex stratified retinal tissue. Yet, ROs lack vascularization and cannot recapitulate the important physiological interactions of matured photoreceptors and the retinal pigment epithelium (RPE). In this study, we present the retina-on-a-chip (RoC), a novel microphysiological model of the human retina integrating more than seven different essential retinal cell types derived from hiPSCs in a vasculature-like perfusion and enabling, for the first time, the recapitulation of the interaction of mature photoreceptor segments with RPE in vitro. We show that this interaction enhances the formation of outer segment like-structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics. In addition, we demonstrate the applicability of the RoC for drug testing, by reproducing the retinopathic side effects of the anti-malaria drug chloroquine and the antibiotic gentamicin. The developed hiPSC-based RoC has the potential to promote drug development and provide new insights into the underlying pathology of retinal diseases.

Human induced pluripotent stem cell (hiPSC)-derived organoids mimicking tissues and organs in vitro have advanced medical research, as they opened up new possibilities for in-depth basic research on human organ development as well as providing a human in vitro model for personalized therapeutic approaches. hiPSC-derived retinal organoids have proven to be of great value for modeling the human retina featuring a very similar cellular composition, layering, and functionality. The technically challenging imaging of three-dimensional structures such as retinal organoids has, however, raised the need for robust whole-organoid imaging techniques. To improve imaging of retinal organoids we optimized a passive clearing technique (PACT), which enables high-resolution visualization of fragile intra-tissue structures. Using cleared retinal organoids, we could greatly enhance the antibody labeling efficiency and depth of imaging at high resolution, thereby improving the three-dimensional microscopy output. In that course, we were able to identify the spatial morphological shape and organization of, e.g., photoreceptor cells and bipolar cell layers. Moreover, we used the synaptic protein CtBP2/Ribeye to visualize the interconnection points of photoreceptor and bipolar cells forming the retinal-specific ribbon synapses.

In recent years, organ-on-chip (OoC) systems have provoked increasing interest among researchers from different disciplines. OoCs enable the recreation of in vivo-like microenvironments and the generation of a wide range of different tissues or organs in a miniaturized way. Most commonly, OoC platforms are based on microfluidic modules made of polydimethylsiloxane (PDMS). While advantageous in terms of biocompatibility, oxygen permeability, and fast prototyping amenability, PDMS features a major limitation as it absorbs small hydrophobic molecules, including many types of test compounds, hormones, and cytokines. Another common feature of OoC systems is the integration of membranes (i) to separate different tissue compartments, (ii) to confine convective perfusion to media channels, and/or (iii) to provide mechanical support for cell monolayers. Typically, porous polymer membranes are microstructured using track-etching (e.g., polyethylene terephthalate; PET) or lithography (e.g., PDMS). Although membranes of different biomechanical properties (rigid PET to elastic PDMS) have been utilized, the membrane structure and material remain mostly artificial and do not resemble in vivo conditions (extracellular matrix). Here, we report a method for the reliable fabrication and integration of electrospun membranes in OoC modules, which are made of laser-structured poly­(methyl methacrylate) (PMMA). The choice of PMMA as base material provides optical parameters and biocompatibility similar to PDMS while avoiding the absorption problem. Using electrospinning for the generation of 3D membranes, microenvironments resembling the native extracellular matrix (ECM) can be generated. We tested two different kinds of electrospun membranes and established processes for a tight integration into PMMA modules. Human (microvasculature) endothelial as well as (retinal pigment) epithelial cell layers could be successfully cultured inside the systems for up to 7 days, while being either directly exposed to (endothelial cells) or protected (epithelial cells) from the shear flow. Our novel method enables the versatile fabrication of OoC platforms that can be tailored to the native environment of tissues of interest and at the same time are applicable for the testing of compounds or chemicals without constraints.

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.