Comparative Analysis of the Physiological and Transport Functions of Various Sources of Renal Proximal Tubule Cells Under Static and Fluidic Conditions in PhysioMimix{trade mark, serif} T12 Platform.

In vitro models that can faithfully replicate critical aspects of kidney tubule function such as directional drug transport are in high demand in pharmacology and toxicology. Accordingly, development and validation of new models is underway. The objective of this study was to characterize physiological and transport functions of various sources of human renal proximal tubule epithelial cells (RPTECs). We tested TERT1-immortalized RPTEC, including OAT1-, OCT2- or OAT3-overexpressing variants, and primary RPTECs. Cells were cultured on transwell membranes in static (24-well transwells) and fluidic (transwells in PhysioMimix{trade mark, serif} T12 organ-on-chip with 2 mL/s flow) conditions. Barrier formation, transport, and gene expression were evaluated. We show that two commercially available primary RPTECs were not suitable for studies of directional transport on transwells because they formed a substandard barrier even though they exhibited higher expression of transporters, especially under flow. TERT1-parent, -OAT1 and -OAT3 cells formed robust barriers, but were unaffected by flow. TERT1-OAT1 cells exhibited inhibitable para-aminohippurate transport, it was enhanced by flow. However, efficient tenofovir secretion and perfluorooctanoic acid reabsorption by TERT1-OAT1 cells were not modulated by flow. Gene expression showed that TERT1 and TERT1-OAT1 cells were most correlated with human kidney than other cell lines, but that flow did not have noticeable effects. Overall, our data show that addition of flow to in vitro studies of the renal proximal tubule may afford benefits in some aspects of modeling kidney function, but that careful consideration of the impact such adaptations would have on the cost and throughput of the experiments is needed. Significance Statement The topic of reproducibility and robustness of the complex microphysiological systems is looming large in the field of biomedical research; therefore, the uptake of these new models by the end-users is slow. This study systematically compared various RPTEC sources and experimental conditions, aiming to identify the level of model complexity needed for testing renal tubule transport. We demonstrate that while tissue chips may afford some benefits, their throughput and complexity need careful consideration in each context of use.

IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin.

Introduction: Hepatotoxicity induced by immunotherapeutics is an appearing cause for immune-mediated drug-induced liver injury. Such immuno-toxic mechanisms are difficult to assess using current preclinical models and the incidence is too low to detect in clinical trials. As hepatotoxicity is a frequent reason for post-authorisation drug withdrawal, there is an urgent need for immuno-inflammatory in vitro models to assess the hepatotoxic potential of immuno-modulatory drug candidates. We developed several immuno-inflammatory hepatotoxicity test systems based on recombinant human interleukin-2 (aldesleukin). Methods: Co-culture models of primary human CD8+ T cells or NK cells with the hepatocyte cell line HepaRG were established and validated with primary human hepatocytes (PHHs). Subsequently, the HepaRG model was refined by increasing complexity by inclusion of monocyte-derived macrophages (MdMs). The main readouts were cytotoxicity, inflammatory mediator release, surface marker expression and specific hepatocyte functions. Results: We identified CD8+ T cells as possible mediators of aldesleukin-mediated hepatotoxicity, with MdMs being implicated in increased aldesleukin-induced inflammatory effects. In co-cultures of CD8+ T cells with MdMs and HepaRG cells, cytotoxicity was induced at intermediate/high aldesleukin concentrations and perforin was upregulated. A pro-inflammatory milieu was created measured by interleukin-6 (IL-6), c-reactive protein (CRP), interferon gamma (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1) increase. NK cells responded to aldesleukin, however, only minor aldesleukin-induced cytotoxic effects were measured in co-cultures. Results obtained with HepaRG cells and with PHHs were comparable, especially regarding cytotoxicity, but high inter-donor variations limited meaningfulness of the PHH model. Discussion: The in vitro test systems developed contribute to the understanding of potential key mechanisms in aldesleukin-mediated hepatotoxicity. In addition, they may aid assessment of immune-mediated hepatotoxicity during the development of novel immunotherapeutics.

Considerations from an International Regulatory and Pharmaceutical Industry (IQ MPS Affiliate) Workshop on the Standardization of Complex In Vitro Models in Drug Development.

In May 2022, there is an International Regulatory and Pharmaceutical Industry (Innovation and Quality [IQ] Microphysiological Systems [MPS] Affiliate) Workshop on the standardization of complex in vitro models (CIVMs) in drug development. This manuscript summarizes the discussions and conclusions of this joint workshop organized and executed by the IQ MPS Affiliate and the United States Food and Drug Administration (FDA). A key objective of the workshop is to facilitate discussions around opportunities and/or needs for standardization of MPS and chart potential pathways to increase model utilization in the context of regulatory decision making. Participation in the workshop included 200 attendees from the FDA, IQ MPS Affiliate, and 26 global regulatory organizations and affiliated parties representing Europe, Japan, and Canada. It is agreed that understanding global perspectives regarding the readiness of CIVM/MPS models for regulatory decision making and potential pathways to gaining acceptance is useful to align on globally. The obstacles are currently too great to develop standards for every context of use (COU). Instead, it is suggested that a more tractable approach may be to think of broadly applicable standards that can be applied regardless of COU and/or organ system. Considerations and next steps for this effort are described.

Reproducibility and Robustness of a Liver Microphysiological System PhysioMimix LC12 under Varying Culture Conditions and Cell Type Combinations.

The liver is one of the key organs for exogenous and endogenous metabolism and is often a target for drug- and chemical-driven toxicity. A wide range of experimental approaches has been established to model and characterize the mechanisms of drug- and chemical-induced hepatotoxicity. A number of microfluidics-enabled in vitro models of the liver have been developed, but the unclear translatability of these platforms has hindered their adoption by the pharmaceutical industry; to achieve wide use for drug and chemical safety evaluation, demonstration of reproducibility and robustness under various contexts of use is required. One of these commercially available platforms is the PhysioMimix LC12, a microfluidic device where cells are seeded into a 3D scaffold that is continuously perfused with recirculating cell culture media to mimic liver sinusoids. Previous studies demonstrated this model's functionality and potential applicability to preclinical drug development. However, to gain confidence in PhysioMimix LC12's robustness and reproducibility, supplementary characterization steps are needed, including the assessment of various human hepatocyte sources, contribution of non-parenchymal cells (NPCs), and comparison to other models. In this study, we performed replicate studies averaging 14 days with either primary human hepatocytes (PHHs) or induced pluripotent stem cell (iPSC)-derived hepatocytes, with and without NPCs. Albumin and urea secretion, lactate dehydrogenase, CYP3A4 activity, and metabolism were evaluated to assess basal function and metabolic capacity. Model performance was characterized by different cell combinations under intra- and inter-experimental replication and compared to multi-well plates and other liver platforms. PhysioMimix LC12 demonstrated the highest metabolic function with PHHs, with or without THP-1 or Kupffer cells, for up to 10-14 days. iPSC-derived hepatocytes and PHHs co-cultured with additional NPCs demonstrated sub-optimal performance. Power analyses based on replicate experiments and different contexts of use will inform future study designs due to the limited throughput and high cell demand. Overall, this study describes a workflow for independent testing of a complex microphysiological system for specific contexts of use, which may increase end-user adoption in drug development.

The evolving role of investigative toxicology in the pharmaceutical industry.

For decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies.

Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.

Analysis of reproducibility and robustness of OrganoPlate® 2-lane 96, a liver microphysiological system for studies of pharmacokinetics and toxicological assessment of drugs.

Establishing the functionality, reproducibility, robustness, and reliability of microphysiological systems is a critical need for adoption of these technologies. A high throughput microphysiological system for liver studies was recently proposed in which induced pluripotent stem cell-derived hepatocytes (iHeps) and non-parenchymal cells (endothelial cells and THP-1 cells differentiated with phorbol 12-myristate 13-acetate into macrophage-like cells) were co-cultured in OrganoPlate® 2-lane 96 devices. The goal of this study was to evaluate this platform using additional cell types and conditions and characterize its utility and reproducibility. Primary human hepatocytes or iHeps, with and without non-parenchymal cells, were cultured for up to 17 days. Image-based cell viability, albumin and urea secretion into culture media, CYP3A4 activity and drug metabolism were assessed. The iHeps co-cultured with non-parenchymal cells demonstrated stable cell viability and function up to 17 days; however, variability was appreciable both within and among studies. The iHeps in monoculture did not form clusters and lost viability and function over time. The primary human hepatocytes in monoculture also exhibited low cell viability and hepatic function. Metabolism of various drugs was most efficient when iHeps were co-cultured with non-parenchymal cells. Overall, we found that the OrganoPlate® 2-lane 96 device, when used with iHeps and non-parenchymal cells, is a functional liver microphysiological model; however, the high-throughput nature of this model is somewhat dampened by the need for replicates to compensate for high variability.

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.

An innovative study design with intermittent dosing to generate a GLP-regulatory package in preclinical species for long lasting molecule M5717, inhibitor of Plasmodium eukaryotic translation elongation factor 2.

M5717 is a novel drug inhibiting synthesis of elongation factor 2 (PeEF2) in Plasmodium species, showing potent anti-malarial activity in preclinical studies. Traditional daily-dosing animal experiments estimating maximum safe starting dose for a first-in-human study ('no observed adverse effect level'; NOAEL) were unsuccessful due to the long pharmacokinetic half-life of M5717, causing significant drug accumulation and high exposure. This study describes an innovative strategy to produce a GLP-certified toxicology package and estimate NOAEL for long-lasting molecules like M5717. Simulated pharmacokinetic/toxicokinetic profiles were used to design the dosing schedule for preclinical safety studies and to determine the 14-day total exposure. Animals (rats/dogs) were administered various doses of M5717 using an intermittent dosing schedule allowing partial drug elimination and alleviation of toxicity during off-treatment days to maintain a minimal parasitical concentration (MPC) of 10 ng/mL; subsequently animals were monitored for toxicity and mortality. Results showed good correlation to the modelled data used to design the dosing regimen and required MPC was reached for M5717 in study animals and could be used to calculate NOAEL. This fit-for-purpose study design allowed for maintaining clinically relevant exposure to M5717, whilst minimizing toxicity-causing compound accumulation, an aspect unaddressed by traditional NOAEL-estimating experiments. This is the first time that a compound-specific, species-specific, kinetic model-based approach to preclinical study design for regulatory toxicology studies has been described and applied to an antimalarial drug candidate with long pharmacokinetic half-life. It has potential for application to other drugs with long half-lives, supporting their clinical development.

Human Retinal Organoids Provide a Suitable Tool for Toxicological Investigations: A Comprehensive Validation Using Drugs and Compounds Affecting the Retina.

Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies.

Structure-Based Optimization and Discovery of M3258, a Specific Inhibitor of the Immunoproteasome Subunit LMP7 (ß5i).

Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (ß1, ß2, and ß5). LMP7 (ß5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.

Deconvolution of Cytochrome P450 Induction Mechanisms in HepaRG Nuclear Hormone Receptor Knockout Cells.

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and PXR/CAR knockout (KO) HepaRG cells, as well as a PXR reporter gene assay, were used to investigate the mechanism of CYP3A4 and CYP2B6 induction by prototypical substrates and a group of compounds from the Merck KGaA oncology drug discovery pipeline. The basal and inducible gene expression of CYP3A4 and CYP2B6 of nuclear hormone receptor (NHR) KO HepaRG relative to control HepaRG was characterized. The basal expression of CYP3A4 was markedly higher in the PXR (10-fold) and CAR (11-fold) KO cell lines compared with control HepaRG, whereas inducibility was substantially lower. Inversely, basal expression of CYP3A4 in PXR/CAR double KO (dKO) was low (10-fold reduction). Basal CYP2B6 expression was high in PXR KO (9-fold) cells which showed low inducibility, whereas the basal expression remained unchanged in CAR and dKO cell lines compared with control cells. Most of the test compounds induced CYP3A4 and CYP2B6 via PXR and, to a lesser extent, via CAR. Furthermore, other non-NHR-driven induction mechanisms were implicated, either alone or in addition to NHRs. Notably, 5 of the 16 compounds (31%) that were PXR inducers in HepaRG did not activate PXR in the reporter gene assay, illustrating the limitations of this system. This study indicates that HepaRG is a highly sensitive system fit for early screening of cytochrome P450 (P450) induction in drug discovery. Furthermore, it shows the applicability of HepaRG NHR KO cells as tools to deconvolute mechanisms of P450 induction using novel compounds representative for oncology drug discovery. SIGNIFICANCE STATEMENT: This work describes the identification of induction mechanisms of CYP3A4 and CYP2B6 for an assembly of oncology drug candidates using HepaRG nuclear hormone receptor knockout and displays its advantages compared to a pregnane X receptor reporter gene assay. With this study, risk assessment of drug candidates in early drug development can be improved.

Erratum to Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Further, the reference to an article by Bal-Price et al. (2015) should have the following doi:10.1007/s00204-015-1464-2 .

Liver microphysiological systems development guidelines for safety risk assessment in the pharmaceutical industry.

The liver is critical to consider during drug development because of its central role in the handling of xenobiotics, a process which often leads to localized and/or downstream tissue injury. Our ability to predict human clinical safety outcomes with animal testing is limited due to species differences in drug metabolism and disposition, while traditional human in vitro liver models often lack the necessary in vivo physiological fidelity. To address this, increasing numbers of liver microphysiological systems (MPS) are being developed, however the inconsistency in their optimization and characterization often leads to models that do not possess critical levels of baseline performance that is required for many pharmaceutical industry applications. Herein we provide a guidance on best approaches to benchmark liver MPS based on 3 stages of characterization that includes key performance metrics and a 20 compound safety test set. Additionally, we give an overview of frequently used liver injury safety assays, describe the ideal MPS model, and provide a perspective on currently best suited MPS contexts of use. This pharmaceutical industry guidance has been written to help MPS developers and end users identify what could be the most valuable models for safety risk assessment.

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models.

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI.

Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.

Optimizing drug discovery by Investigative Toxicology: Current and future trends.

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

Human-Induced Pluripotent Stem Cells Generate Light Responsive Retinal Organoids with Variable and Nutrient-Dependent Efficiency.

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human-induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal-pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. Stem Cells 2018;36:1535-1551.