Human Retina-on-a-Chip: Merging Organoid and Organ-on-a-Chip Technology to Generate Complex Multi-Layer Tissue Models

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.


Introduction
Retinal diseases such as Stargardt disease, age-related macular degeneration, diabetic retinopathies or retinitis pigmentosa are amongst the leading causes of vision loss in humans [1,2]. Unfortunate for patients suffering from those diseases, there are currently no cures available [3,4]. Moreover, the complex neuro-retinal organization and the vast blood supply make retinal tissue susceptible for side effects of compounds delivered intravitreally or systemically [5,6]. Retinal toxicities are a major issue for a wide range of therapeutic substances and especially for targeted anticancer agents since many of the targets are also expressed in ocular tissues [6]. Although animal models that are used to explore new therapeutic options and assess retinal toxicities resemble the human (patho-)physiology of vision in certain aspects, they fail to reflect fundamental characteristics including trichromacy or a fovea centralis, responsible for high visual acuity (Fig 1). In vitro cell culture assays, on the other hand, are typically based on non-physiological 2D cell cultures, which cannot reflect the complex architecture and cell-cell interactions as well as the blood perfusion. More complex approaches such as retinal explants from human donors provide a full featured model; however, the limited availability and culturability as well as inter-donor variabilities make it unsuited for drug development and testing. The invention of physiologically relevant in vitro models capable of mimicking the human retinal biology is hence of crucial importance. Recent progress in the generation of 3-dimensional (3D) organoids derived from human pluripotent stem cells (hPSC) (derived from both induced (hiPSC) as well as embryonic (hESC) stem cells) enabled the reflection of distinct types of tissues, such as subsystems of the central nervous system including the retina. Retinal organoids (ROs), also called "eyes in the dish" resemble rudimentary optic vesicle-like structures with a retinal layering similar to in vivo conditions [5,6]. These ROs contain most relevant retinal cell types in a physiological layering such as ganglion cells, amacrine cells, horizontal cells, bipolar cells, Müller glia as well as rods and cones. Nevertheless, hPSC-ROs are still facing a variety of drawbacks limiting predictive research on e.g. human retinal development, function or drug response. Some of the major hurdles are (i) the functional maturation of differentiated cells, (ii) lack of essential cell types (e.g. microglia), (iii) lack of a physiological interplay of the various retinal cell types especially of photoreceptors and retinal pigment epithelia (RPE), as well as (iv) a missing vascularization (reviewed in [9,10]). Due to the lack of a physiological perfusion, the delivery of compounds to ROs is uncontrolled and entirely artificial. Here, general limitations of static cell culture apply including non-physiological cell-to-media ratio, uncontrolled shear forces during media exchanges, as well as highly variable conditions between media exchanges. In recent years, the short comings of conventional static cell culture has led to the emergence of microphysiological systems (MPS), specifically Organ-on-a-Chip (OoC) platforms. MPSs have evolved to a powerful alternative for classical cell culture and animal models by providing physiological microenvironments embedded in a vascular-like microfluidic perfusion [11,12]. This new and 4 promising technology has the potential to revolutionize drug development and usher into a new era of personalized medicine. Over the past years, a variety of MPSs have been developed, mimicking, for instance, cardiac [7], lung [15], renal [16], and hepatic tissue [11]. In the context of ophthalmologic research, a variety of approaches have been introduced that represent partial layers of the cornea [9] or the retina [20 -24]. So far, however, no MPS has been able to successfully recapitulate the complex 3D architecture of the human retina. In this study, we developed a physiologically relevant 3D in vitro model of the human retina by combining hiPSC-ROs with hiPSC-derived RPE in a retina-on-a-chip (RoC). This novel microphysiological 5 platform enables enhanced inner and outer segment formation and preservation, a direct interplay between RPE and photoreceptors as well as a precisely controllable vasculature-like perfusion. In order to provide a high-content platform for basic and applied research, we established a toolbox comprising in situ analysis approaches as well as terminal endpoints enabling the monitoring of functionality as well as molecular mechanisms. To demonstrate the applicability for drug screening, the system was exposed to the drugs chloroquine and gentamicin, which are known to have retinopathic side effects [25 -29].

Retinal organoids show rod and cone diversity and simple inner and outer segment formation
ROs derived from hiPSCs harbor all known major retinal subtypes such as ganglion cells, bipolar cells, horizontal cells, amacrine cells, Müller glia and photoreceptors ( [30], Fig 2, S1). Using immunostaining and mRNA qPCR, the presence of the retinal cell types as well as crucial retinal morphological cues such as inner and outer photoreceptor segment formation, a tightly formed outer limiting membrane (OLM) and a correct layering was verified (Fig 2, S1). In order to reach a suitable maturation, ROs were differentiated for 180 days. ROs of that age harbor matured photoreceptors which have simple forms of inner and outer segments, situated on the surface of the ROs which can be seen in bright field microscopy and by immunostaining of respective markers (Fig 2b-f). Immunostaining of the respective organoids demonstrate the presence of a mixed population of rods and cones, identified by specific markers (rhodopsin for rods and Arrestin-3 for cones, Fig 2c-e, S1). In order to analyze segment formation, we used ROM1 as it has been previously shown as specific outer segment marker [31] and verified the segment specificity by co-staining with the rod marker rhodopsin (Fig 2c-d, f). Further, we tested the previously described protein PNA Lectin, which was delineated to specifically bind to cone photoreceptor segments [32]. Co-staining of rhodopsin with PNA lectin exhibited that not only cone but also rod segments in ROs are labelled with PNA lectin (Fig 2e).

Microphysiological Retina-on-a-Chip
To recapitulate the complex in vivo anatomy of the human retina in vitro (Fig 3a), we developed a microfluidic platform that enables the culture of hiPSC-derived RPEs and ROs in a defined physiological structure (Fig 2b). Each RoC features four identical micro-tissues connected via a microchannel and is comprised of two transparent and biocompatible polymer layers. The top layer features the compartments for the ROs and RPE, whereas the bottom layer provides a channel for a vasculaturelike perfusion enabling a constant supply of nutrients and compounds. Both layers are separated by a thin porous membrane mimicking the endothelial barrier and shielding the tissues from shear forces while simultaneously enabling the exchange of nutrients and metabolites (Fig 3b). The tissue compartments are accessible from above for the initial seeding process and sealed during the subsequent culture process to avoid evaporation and contamination. A stable tissue comprising ROs and RPE is achieved by first seeding hiPSC-derived RPE cells at a defined density into each tissue compartment (Fig 3c) and subsequent culture for 24 hours (Fig 3d). This step was followed by injection of ROs embedded in a hyaluronic acid-based hydrogel (representing the major component of the interphotoreceptor matrix between REPE and PRC) into the tissue compartments (Fig 3e). This led to the formation of a thin hydrogel layer, generating a defined distance between RPE cells and outer limiting membrane of the ROs. Thereby, a direct contact and, thus, an uncontrolled outgrowth of cells from the ROs during culture was successfully avoided. ROs and RPE were cultured in the system for at least 3 days prior to further functionality assessment or experimentation.

Specific marker expression and polarization of retinal pigment epithelial cells in the RoC
A polarized and functional RPE is crucial for the survival of photoreceptors in vivo and a vital part of the visual cycle shuttling retinoic acid between RPE and photoreceptor outer segment [33,34]. Therefore, the RPE in the RoC was thoroughly tested for its marker expression and polarization (Fig 4,   S2). Expression of RPE markers PAX6 and MITF can be observed after 7-14 days in the chip (Fig 4a-b).
Mature RPE inside the chip displayed cobble stone-like morphology and tight junction formation visualized by ZO-1 staining (Fig 4c). The melanosome and pigmentation marker Melanoma gp100 (also called PMEL17 or Silver locus protein) [35,36] was highly expressed in chip-cultured RPE (Fig 4d) indicating a strong pigmentation. A conclusive evidence for the maturation and proper functionality of RPE is its state of polarization [34,37]. Electron microscopy analysis revealed not only the strong pigmentation of the RPE but also the presence of apical microvilli as well as a basal membrane already 10 after 7 days of on-chip culture (Fig 4e). Further, we showed the polarized expression of ezrin, an apical microvilli marker [38] (Fig 4f). Finally, polarized RPE displayed basal secretion of VEGF-A, which could be measured on-chip by using a double-channel chip in which basal and apical medium could be collected separately (Fig 4g). The VEGF-A concentration was higher in the basal channel than in the apical (70 vs 40 pg/chip in 24 hours). Taken together, the RPE in the RoC is strongly pigmented and polarized, and expresses RPE markers.

Enhanced outer segment formation in the RoC
The close proximity and the precisely orchestrated interaction of photoreceptors and the RPE layer is fundamental for vision, ensuring the phagocytosis and processing of shed photoreceptor outer segments (POS) as well as a supply of nutrients and oxygen [25]. The RoC device allows the establishment of a defined interaction site between the segment structures of the RO and RPE cells without impairing neither structure nor viability of the organoid (Fig S3-4). Live cell imaging in the chip was enabled by transducing RPE cells with an IRBP-GFP viral vector and by marking the surface structures of the organoids with PNA lectin coupled to Alexa Fluor® 568 prior to on-chip culture (Fig   5a). By measuring the distance between lectin-marked segment tips and GFP-labelled RPE, we found a distance of approximately 5 µm ± 3.19 µm over different experiments (Fig 5c). Subsequently, immunostaining using rhodopsin (rod outer segments) and phalloidin (cytoskeleton of the RPE and the RO including tight junctions) revealed that the segment structures and RPE cells are in close apposition on-chip (Fig 5b).
To further study the mechanically delicate interaction site between ROs and RPE in cryosections, we performed immunofluorescence analysis using a specifically tailored chip version (Fig 5d). After 7 days of on-chip culture, the close proximity of RPE and RO was preserved and no indication of cell outgrowth or general loss of integrity of the continuous OLM, labeled by the actin-cytoskeleton marker phalloidin, was observed (Fig 5d). Further, the hydrogel-filled space between the RPE monolayer and the RO was strongly invaded by rhodopsin and ROM1 (outer segment marker)-positive clusters indicating an increased and cumulated presence of inner and outer segment-like structures (Fig 5d). A detailed analysis of the interaction site via electron microscopy confirmed the formation of numerous inner segments with clusters of mitochondria as well as the maintenance of the OLM (Fig 5e). The distance of outer segment tips and RPE microvilli in this exemplary image is around 5 µm (Fig 5e), which is in accordance with the data shown above.
Next, we examined whether the formation and preservation of outer segment-like structures is improved in the RoC in comparison to a RO cultured in the chip without RPE and conventional dish cultured ROs (Fig 5f). Using electron microscopy, we could find outer segment-like structures in all conditions (Fig 5f), displaying distinct disk formation (Fig 5g, exemplarily). However, in the RoC system (RoC), the number of outer segment structures was about three times higher than in RO chips without RPE (RoC w/o RPE) and in dish cultured ROs (Fig 5h). Interestingly, there was no difference observed in the RoC chip without RPE and dish culture, indicating a positive effect of the RPE on the outer segment formation and preservation. In summary, the RoC increases the formation of outer segmentlike structures on the RO without disturbing the normal survival and makeup of the organoid structure.

Modeling key functionalities of the visual cycle
To assess whether the ROs and the RPE can reproduce principle retinal functionality on-chip, we first assessed the ability of the RO photoreceptors to produce an in vivo like calcium flux. Calcium ions (Ca2+) are fundamentally important for the function of photoreceptors and involved in many processes ranging from photodetection, transduction and synaptic transfer (reviewed in [25]). To be able to monitor calcium transients and investigate photoreceptor metabolism and functionality, we established on-chip calcium imaging as an easy to perform read-out method. By loading RO and RPE on-chip with the calcium dye fura-2-AM, we were able to image calcium dynamics for individual photoreceptors or RPE cell ROIs over an extended timespan (Fig S5).
Second, we focused on one of the main functions of RPE, the phagocytosis of substances and cell remnants in form of membrane stacks produced by the photoreceptors. The general capability of the hiPSC-RPE to perform phagocytosis was initially verified using bovine retinal outer segments (Fig S6).
In the RoC platform, RPE cells were labelled by a promoter-driven GFP to mark cell bodies. By labelling the organoids with PNA lectin before the RoC was set up, segment structures were labelled and visualized (as described in Fig 5a). Already after 1 day in culture, PNA lectin-positive structures were found within the RPE cell bodies, indicating ongoing digestion of segment particles (Fig 6a). The composition of these particles was examined by immunostaining for rhodopsin (Fig 6b). This revealed that many of the lectin-positive particles found in the RPE cells were positive for rhodopsin. Next, we asked whether the particles taken up by the RPE are found in the early endosomes, which, in a later step, fuse to phagolysosomes for a full digestion. We labelled RPE cells prior to setting up the RoC with a GFP construct visualizing early endosome complexes (Fig 6c, red). After on-chip immunostaining of 14 rhodopsin, specific co-localization of GFP-labelled early endosomes with rhodopsin-positive fragments was detected (Fig 6c).
Finally, we visualized the RPE endosomes in the RoC using electron microscopy. We examined day 7 RoCs, identifying indigested outer segment-like structures in the RPE (Fig 6d). Here, we found several membrane stack-structures in the RPE below the RO. These structures displayed multi-membrane formation (red arrow) as well as small round membrane structures, both strongly reminiscent of similar features found in outer segment-like structures in ROs (Fig 6d, right). Taken together, this strongly indicates a functional indigestion of segment structures by the RPE, which is a major prerequisite for a functional visual cycle and therefore a physiological RPE-photoreceptor model.

Evaluation of drug-induced retinopathy
In order to highlight the RoC's applicability for drug development and toxicology assessment, we exposed the system to the anti-malaria drug chloroquine (CQ) and the antibiotic gentamicin (GM), which both were previously shown to have pathological side effects on the retina [25 -29].
After 3 days of on-chip culture, retinal tissue was exposed to two different concentrations of CQ (20 µg/ml and 80 µg/ml) for 3 additional days. Concentrations were chosen based on previously described effects of CQ on cell viability using the RPE cell line ARPE-19 [39], and preliminary experiments using hiPSC-RPE for CQ treatment (Fig S7). Subsequent to the treatment, the RoCs were stained with propidium iodide (PI) to assess cell death (Fig 7a). Additionally, they were co-stained with the lysosomal marker protein LAMP2 (Fig 7c) since lysosomal dysfunction is involved in the pathophysiology of CQ [39 -41]. When RoCs were exposed to 20 µg/ml CQ, no significant impact on cell viability (Fig 7b) and only a minor increase in LAMP2 signal (Fig 7c) were observed. However, at a concentration of 80 µg/ml, cell viability was clearly impacted as shown by a significantly stronger PI staining (Fig 7a,b) compared to controls without CQ treatment. Furthermore, after exposure to 80 µg/ml CQ, a strong LAMP2 signal was visible (Fig 7c), indicating an enlargement of lysosomes where the drug is accumulating and leading to lysosomal dysfunction. The increase in LAMP2 was not limited to the RPE but was also very pronounced in the RO (Fig 7c).
To assess the effect of GM, the antibiotic was added for 6 days to the RoC and to RoC without RPE (Fig   7d,e). In RoCs without RPE, a significant increase in cell death was observed at a GM concentration of 16 0.5 mg/ml (Fig 7f), further increased in RoCs exposed to a 5-fold higher GM concentration (2.5 mg/ml).
In the complete RoC (RO and RPE), similar effects became apparent: Both, low and high GM concentrations led to a significant increase in PI signal (Fig 7g). Since the quantified PI signal was a combined signal of RPE and RO cell death, we asked whether there the RO was affected differently by the drug when comparing the conditions with and without RPE (RoC and RoC w/o RPE). For that purpose, we substracted the PI signal localized in the RPE from the calculated values of the whole RoC in the 0.5 mg/ml treated chips (Fig 7h). Interestingly, we found a significant increase of PI in the condition without RPE, but an unchanged PI signal in the RoC-cultured RO. This is in contrast to the results from the RoC without RPE where a robust increase was observed when treating the chip with 0.5 mg/Ml GM (Fig 7h). This could indicate a barrier or even protective function of the RPE, shielding the organoid from the drug and decreasing the toxic effect of the drug on the organoid. ROs solved this issue partially [30], but were still limited due to the absence of a functional interaction between RPE and photoreceptor outer segments (POS). Attempts in recreating the retinal niche in the 20 past have failed to recapitulate the precise RPE-PR arrangement, and thus, did not yield matured photoreceptors with large formations of membrane disk-containing outer segments. The RoC platform is able to mimic this particular niche and a physiological interaction of RPE and POS, by embedding ROs and RPE in hyaluronic-based hydrogel in specifically tailored microfluidic chambers. This arrangement is much more physiological and controlled than previous approaches employing an unpredictable and unorganized RPE formation during RO generation. This inter-photoreceptor matrix in the RoC provides mechanical stability for the small and fragile developing POS, which would otherwise either be damaged or prevented from outgrowth as in conventional suspension cultures. In the RoC, hence, numerous outer segments facing towards the RPE were formed. The flexible and highly controlled tissue assembly paves the way for the modelling of a variety of disease states, e.g. by combining ROs derived from patients suffering from retinal diseases with RPE derived from the same or healthy donors.
Post-mortem human retinal explants are the sole human models that are currently available and achieve a comparable level of complexity. Those ex-vivo models are, however, greatly limited in terms of supply, with respect to long-term culture, and due to inter-patient variability. Moreover, they are not applicable for studies targeting developmental aspects. The introduced RoC systems is based entirely on hiPSCs that are easily expandable and cryo-conservable. This not only avoids the problem of limited supply but also provides the capability to create a multitude of genetically identical systems and sets the foundation for a scale-up to higher throughput systems, provided an appropriate framework and an automated process landscape is established [42]. The hiPSC technology further paves the way for the generation of disease-specific as well as patient-specific models opening up future applications in disease modeling and personalized medicine. Additionally, hiPSC derived ROs exhibit the ability to stay viable for more than a year in vitro. This is a crucial aspect in terms of answering developmental aspects, studying disease initialization and progression as well as assessing long-term effects or side effects of drugs.
Both current in vitro and ex vivo models share one major limitation, the lack of a vascularization or vasculature-like perfusion. This aspect of the microfluidic RoC adds a further dimension of physiological relevance and advantage over the conventional models as described above.
Animal models are so far the only model systems that feature a blood circulation as well as a structural tissue complexity. Yet, besides issues of ethical concerns, results from animal models are often not translatable to humans as none of the small animals used in the field of retina research is able to fully represent the human retinal system. The human genetic background and recapitulation of human retinal tissue structure, hence, provide the potential for the RoC model to overcome those translation hurdles. Animal models, however, still possess structural elements of the visual systems, i.e. optical nerve and inner blood-retinal barrier, which cannot be re-created in vitro, so far.
Taken together, the introduced RoC represents a highly advanced in vitro model, which is not hampered by many of the limitations of conventional (in vitro, ex vivo, in vivo) model systems and which can be the first step towards the reduction and replacement of animal models in the field of ophthalmology. Further development will target challenges of innervation (addition of an optical nerve), incorporation of blood-retinal barriers, the integration into multi-organ platforms, as well as the generation of disease-specific systems.

Summary
The scarce availability of physiological relevant in vitro models of the human retina and the limited capability of animal models to recapitulate physiological human responses has i) hampered the development of potential new drugs to treat degenerative diseases such as Stargardt disease, age-

Competing interests
The authors declare no competing interests.

Fabrication of Retina MPS
The RoC consists of two Polydimethylsiloxane (PDMS) layers and a porous PET membrane in between, bonded to a thin glass slide (170 µm). First, PDMS master molds were fabricated. For the media channel mold, SU8-50 photoresist (MicroChem, USA) was spin-coated onto a previously cleaned 4" silicon wafer to obtain a height of 100 µm. To create the desired structure, the substrate was exposed to 350 mJ/cm 2 of UV light, followed by development in SU-8 developer (micro resisttechnology GmbH, Germany) for 6 minutes. Finally, the wafer was rinsed with isopropanol and blow-dried using nitrogen.
The second wafer for RO and RPE culture was fabricated in two steps. Initially, a base layer of 25 µm for the membrane insert was fabricated by spin-coating a first layer of photoresist SU8-. The exposure to UV light in this case was 200 mJ/cm 2 . Subsequently, the wafer was developed in SU-8 developer for 4 minutes, rinsed in isopropanol and blow-dried with nitrogen. Next, the wafer was coated with a second layer of SU8-3025 to fabricate the tissue channels with a height of 40 µm. The wafer was exposed to UV light at 250 mJ/cm 2 for 10 seconds and developed for 4 minutes. Afterwards, both master molds were silanized with chlorotrimethylsilane (Sigma-Aldrich, Germany). Subsequently, Sylgard 184 PDMS (Dow Corning, USA) was mixed at a 10:1 ratio of prepolymer to curing agent and molded by using the wafers as negative master mold. The layer for the media supply was made by exclusion molding followed by curing overnight at 60°C. The RO/RPE culture layer was fabricated by pouring 25 g of the PDMS mixture onto the master mold and curing it overnight at 60°C. Next, the PDMS slabs were peeled off the wafers and the media-supply layers were bonded to a glass slide previously cleaned by a 30-second exposure to oxygen plasma at 50 Watts. Inlets and outlets were punched using a biopsy puncher with a diameter of 0.75 mm. To culture the cells and organoids, four chambers were punched out of the PDMS with a biopsy puncher of 2 mm diameter. Semipermeable membranes with an diameter of 20 mm, made from PET (Sabeu GmbH, Germany) with a pore diameter of 3 µm and a thickness of 10-20 µm, were functionalized using bis-[3-trimethoxysilypropyl]amine (Sigma-Aldrich, Germany). Before assembly, both PDMS layers were cleaned with isopropanol and Scotch tape to remove dust particles. Afterwards, both layers were treated with an oxygen plasma at 50 W for 30 s. Then, the membrane was placed into the inlay of the RO/RPE culture layer. Finally, both layers were aligned to each other using a stereo microscope and baked overnight at 60°C to stabilize bonding.

Cell culture iPSC Culture
All hiPSC cell lines were derived from healthy donors as previously described [43] and tested for stem cell markers and germ-layer differentiation potential. hiPSCs were cultured on Matrigel (hESCqualified, BD Biosciences, USA)-coated plates with FTDA medium [44]. Cells were passaged every 6-7 days using Dispase (Stemcell Technolgies, Canada). Differentiated colonies were removed manually by scraping. All procedures were in accordance with the Helsinki convention and approved by the Ethical From day 70-100, BRDM with FBS and taurine was further supplemented with 1 µM retinoic acid (Sigma-Aldrich, USA), which was reduced to 0.5 µM during days 100-190 and removed afterwards.

Differentiation of retinal pigment epithelial cells
RPE cells were derived as a product from RO differentiation following (slightly adapted) procedures of Zhong et al. and Ohlemacher et al. [8] [45]. For this purpose, pigmented areas or spheres were removed from ROs using microscissors under an inverted microscope. The pigmented areas were collected in 1.5 ml Eppendorf tubes (Eppendorf, Germany) and washed once with Dulbecco's phosphate-buffered saline (PBS, no calcium, no magnesium, Thermo Fisher Scientific, USA). To dissociate the RPE into single cells for adhesion culture, the pigmented spheres were treated with Accumax (Sigma-Aldrich, USA) for 90 minutes at 37°C and 5% CO 2 and resuspended every 30 minutes using a 100 µl pipette. The reaction was stopped using BRDM with 10% FBS followed by centrifugation at 1500 rpm for 2 minutes. The

Transduction of RPE cells
To generate green fluorescent iPSC-RPE lines, adherent RPE cultures were incubated with lentiviral particles generated from pJG-IRPB-eGFP plasmids [46] (Gift from Deepak Lamba, Thomas Reh) in BRDM + 10% FBS for one day, washed three times with PBS and further cultivated in BRDM.

RoC culture
Individual systems were sterilized via oxygen plasma treatment for 3 minutes at 50 Watts and placed into PBS-filled 50 ml tubes to displace the air in the channels. Before seeding hiPSC-RPE cells into the MPS, each system was removed from the tube, carefully dried with a paper towel and placed into a 10 cm dish. Each well was coated for 2 hours with 50 µg/ml Laminin in DMEM/F12 at 37°C and 5% CO2. RPE cells were detached and dissociated using Accumax at 37°C and 5% CO 2 for 10-40 minutes, depending on the adherence and passage of the cells. To remove cell agglomerates, a 70 µm cell strainer was used. As a next step, each well was seeded with RPE at a density of 27 000 cells in a volume of 4.5 µl BRDM supplemented with 10% FBS. RoC were incubated for at least 2 hours at 37°C and 5% CO 2 to allow RPE cells to adhere to the semipermeable membrane. The medium was changed every day for 1-3 days prior ROs were loaded into the RoCs. ROs were placed onto the RPE covered membrane. Hyaluronic acid-based hydrogel HyStem-C (ESI Bio, USA) was prepared according to the 26 manual and added to the well by pipetting. During culture, the chambers were covered by a sterile adhesive tape (optical adhesive covers, Thermo Fischer Scientific, USA) to avoid evaporation. BRDM supplemented with 100 µM taurine and 10% FBS was supplied at a constant flow rate of 20 µl/h by syringe pump.

Drug Treatment
RPE and ROs were seeded into the RoC as described above. Subsequently, ROs and RPE were either treated for three days with 20 and 80 μg/ml chloroquine (Sigma-Aldrich, USA) in BRDM using a syringe pump at a flow rate of 30 µl/h or with 0.5 mg/ml and 2 mg/ml Gentamicin (Sigma-Aldrich, USA) for 6 days. For every treatment, control RoCs were also used, without addition of equal amounts of the solvent (H 2 O). After 3 days of treatment, cells in the RoCs were stained using HOECHST (Thermo Fischer Scientific, USA) and 3 µM propidium iodide (PI, Sigma Aldrich, USA) to assess cell death. RoCs were washed twice with PBS using a syringe and fixed with 4% PFA for immunhistochemical staining of LAMP2.

Phagocytosis Assay using bovine ROS
Bovine rod outer segments were isolated as previously described [47]. For the phagocytosis assay outof-the-chip, hiPSC-RPE was plated on cover slips after coating with 0.01% Poly-L-Ornithine Solution and Laminin as described above. For the phagocytosis assay in the RPE-chip, RPE was loaded as described above into the Laminin-coated wells. On the next day, hiPSC-RPE on coverslips or in the chip were incubated with bovine photoreceptor outer segments (POS) at a density of 10 POS/RPE in BRDM for 2h at 37°C, then washed with PBS 3x and cultivated for additional 2h in BRDM and then fixed with 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) and 10% sucrose (Carl Roth, Karlsruhe, Germany) in PBS for 20 minutes at room temperature for immunohistochemistry.

ELISA assay VEGF-A secretion
Specialized double-channel RoCs were generated for apical and basal secretion measurement. These chips were identical with the previously described setup, except that an additional channel was included (apical channel) connecting the compartments, with an additional inlet and oulet that allows media flow above the RPE layer, in additional to the media flow below. These double-channel chips were loaded with RPE cells as described and then cultivated for 14 days using a syringe-pump.
The media from the upper and lower-channel outlet were collected in a tube each day and the collected medium was frozen at -20°C. After defrosting, the VEGF-A amount in the conditioned medium of day 14 was measured using a VEGF-A Human ELISA Kit (Thermo Fisher Scientific, USA). The assay was performed according to the manufacturer's protocol and absorbance was read at 450nm.

27
The apical and basal media was analyzed from 3 different chips and initial volumina were noted for calculation of the total substance quantity per chip on the apical or basal side.

Live cell labeling of hiPSC retinal organoids
For live cell labeling of RO photoreceptor segments, ROs were incubated in a reaction tube for 30 minutes in BRDM containing 20 µg/ml PNA lectin-Alexa Fluor® 568 (Thermo Fisher Scientific, USA) or PNA lectin-Alexa Fluor® 647 (Thermo Fisher Scientific, USA) followed by washing with medium four times, prior to the transfer into the RoC.

Live cell endocytosis and phagocytosis assay
For live cell endocytosis experiments, RPE cells were infected overnight with 10 particles/cell of CellLight® Early-Endosomes GFP (BacMam 2.0, Thermo Fisher Scientific, USA) prior to the seeding of RPE into the RoC. Endosome labeling could be detected for >5 days.

Production of agarose RoCs and cryoembedding
Agarose RoCs were produced from an in-house fabricated mold using 4% Agarose/BRDM + 10% FBS containing four separated compartments and a semipermeable membrane (as described in the MPS section) at the bottom of each well. RPE and ROs were loaded into the agarose RoCs as already described. For fixation, agarose RoCs were fixed in 4% paraformaldehyde in 0. for a total of 30 minutes, and postfixed in 1% OsO4 (Electron Microscopy Sciences, Germany) for 1.5 hours at room temperature. After three additional washes in cacodylate buffer and dehydration in 50% ethanol, tissues were counterstained with 6% uranyl acetate dissolved in 70% ethanol (Serva, Heidelberg, Germany) followed by graded ethanol concentrations of ethanol (80% and 96% for 15 minutes each, 100% for two times 10 minutes, acetone 100%, 15 min). The dehydrated samples were incubated in a 2:1 and 1:1 mixture of acetone and Epon resin (Serva, Heidelberg, Germany) for 1 hour each, on a shaker. Finally, organoids were infiltrated with pure Epon and polymerized by overnight incubation at 60°C. The next day, ROs and RPE were punched out of the chambers. Epon punches containing RPE-filter and ROs were embedded in fresh resin in flat molds (Science Services, Germany) and cured 12 hours at 60°C followed by 2 hours at 90°C.
Ultrathin sections (50 nm) were cut on a Reichert Ultracut S (Leica, Germany), collected on copper grids and counterstained with Reynolds lead citrate. Sections were analyzed with a Zeiss EM 900 transmission electron microscope (Zeiss, Germany) equipped with a 2k x 2k CCD camera.
Images were used for quantification of outer segment density using an image analysis software (iTEM, Olympus Soft Imaging Solutions, Germany). To calculate the ratio of outer segments per µm organoid surface, a line was drawn and measured along the outer limiting membrane of the organoid and outer segment structures visible along the line were counted.

Immunohistochemistry
For in situ chip staining, whole-mount staining was performed using a blocking solution of 5% or 10 % normal donkey serum (Millipore, USA) with 0.2% triton-X (Carl Roth, Karlsruhe, Germany) for permeabilization, twice for 1 hour. Primary antibodies were added to the blocking solution for 1 or 2 days at 4°C, then secondary antibodies were added in blocking solution overnight at 4°C. Next, RoC were counterstained with HOECHST 33342 for 10 minutes at room temperature (1:2000, Thermo Fisher Scientific, USA). Washing steps to remove residual antibodies were performed with PBS, three times for 2 hours at room temperature after incubation of primary and secondary antibodies, as well as after HOECHST staining.
Cryosections from agarose-chips and ROs were rehydrated in PBS for 15 minutes and incubated in a blocking solution of 10% normal donkey serum in PBS with 0.2% triton-X for 1 hour. Wholemount ROs were incubated in a blocking solution of 10% normal donkey serum in PBS with 0.2% triton-X for 1 hour. Primary antibodies were diluted in blocking solution and incubated over night at 4°C. Secondary antibodies were diluted in 1:1 blocking solution:PBS and incubated for 2 hours at room temperature.
Mounting was performed with ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific, USA). Washing steps to remove residual antibodies were performed with PBS, three times for 3 minutes at room temperature after primary and secondary antibodies.

TUNEL Assay
TUNEL Assay (Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay; Thermo Fisher Scientific, USA) was performed according to the manufacturer's manual.

Gene expression analysis using Fluidigm qRT-PCR
Total RNA isolation and gene expression analysis was performed as previously described [48]. For quantification of the gene expression of the genes of interest, Taqman® assays were purchased from Thermo Fisher Scientific, USA.
To detect the calcium signals in the RoCs, fluorescent-labeled cells were manually encircled by regions of interest (ROIs) and the obtained ROIs coordinates were used to extract corresponding calcium traces from the ratio-stacks. Average frames of pre-and post-stimulus frames (ImageJ, custom written script) substituted ratio frames during the light stimulation period.

Fluorescence intensity quantification
Fluoresencent signal of propidium iodid signal was quantified using ImageJ (https://imagej.nih.gov/). For signal calculation, fluorescencene signal was quantified before and after PI labelling using ROI selection and mean intensity pixel values. Signal intensities before PI labelling were considered as

Statistical analysis
Two-sided student's t-test was used to analyze differences between samples conditions. Statistical anaylsis was performed with GraphPad Prism 5 (San Diego, California). Data are presented as mean ± S.E.M. p-value is indicated in the respective graphs.

Data availability
The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files.