Supported by the Lowy Medical Research Institute. We would like to thank the Lowy family for their support of the MacTel project. We would like to thank Mari Gantner, Mike Dorrell, and Lea Scheppke for their intellectual input and assistance preparing the manuscript.

<ol>
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Authors have no conflicts of interest.

Differentiation protocol variations 
Since the invention of self-forming optic cups by Yoshiki Sasai&#39;s group20, many labs have developed protocols to generate retinal organoids that can vary at almost every step5,18,19,21. An exhaustive list of protocols can be found in Capowski et al.22. The differentiation protocol we provide here is a simple, low-intervention protocol that provides a good starting point for any lab attempting to differentiate mature retinal organoids. Users are encouraged to explore and adopt variations on this protocol. Common steps for protocol variation are the production and early differentiation of EBs, and the use of differentiation factors such as BMP4, DKK-1, or retinoic acid to improve efficiency5,19,23.
Culturing organoids on a shaker for much of the late-stage development has been an effective step to increase differentiation efficiency and reduce the time spent handling organoids. Bioreactors achieve the same ends24, however, using a shaker with disposable Erlenmyer flasks is more cost-effective and can be done with common lab equipment. The main advantage of keeping the organoids in mobilized suspension is that it removes the need to separate organoids that stick together on the plate while they are growing. The suspended cultures also facilitate greater nutrient exchange, so organoids have a tendency to grow larger than those on a standard still plate. For this reason, it is good to start out by fragmenting plated EBs as small as possible in step 4.1.
Since obtaining mature retinal organoids requires differentiating a single culture for nearly 6 months, performing toxicity assays on retinal organoids can seem time-consuming compared to using a 2-D monoculture. However, toxicity assays are performed on a single control line, and differentiations can be set up routinely. Following an initial 6-month investment, a regular supply of organoids to perform additional experiments and protocol modifications will be at hand without delay. Initiating 2-3 rounds of retinal organoid differentiation every 1-2 months provides ample tissue for complex and thorough sets of experiments.
Organoids provide a versatile model for targeted drug testing. 
This protocol describes a photoreceptor toxicity assay to test the efficacy of lipid-altering drugs to rescue deoxySL-induced cell death. Although this is a specific application showing a pharmacological rescue for a disease-causing agent in MacTel, this protocol can be utilized to test any number of insults (e.g., nutrient deficiency, hypoxic conditions, light toxicity) and pharmacological rescues. Therefore, it can be adapted to model other retinal diseases. In our own work we have shown that by using this same protocol and substituting various related sphingolipid species and drugs that directly block sphingolipid metabolism we were also able to provide insights to the MacTel disease mechanism by determining the specific toxic downstream sphingolipid metabolite that leads to photoreceptor cell death14. Unlike modeling diseases at the level of genetic mutations, which require establishing variant iPSC lines, using organoids to model toxic metabolic conditions and environmental stressors allows for the use of a common control iPSC line as a dynamic model with abundant and readily available tissue source.
The human organoid models are particularly advantageous when studying metabolic diseases in the retina, where different cell types have a unique symbiotic metabolic interplay that cannot be replicated in a monoculture of photoreceptor-like cells4. This complex metabolic model has allowed us to discover the effectiveness of the drug fenofibrate in preventing the toxic effects of deoxySLs directly in humans. In mouse models of elevated deoxySLs14 (unpublished data) and mouse embryonic fibroblast cell culture models of deoxySL metabolism fenofibrate proved to be ineffective16. Testing drugs in the context of complex human tissue models will allow us to identify effective treatments and discount ineffective treatments that would have otherwise been missed had we only relied on mice or simplistic monocultures.
Future development for drug screening 
In this protocol we quantify apoptosis in photoreceptors, the most abundant cell type. However, by utilizing any of the proven antibodies that specifically label the other retinal cell types this protocol is modifiable to assay cell death in any retinal cell type, or cell subtype (e.g., cone vs rod). The disadvantage of quantifying apoptosis using TUNEL staining in tissue slices is that it is a low throughput technique to quantify cell death, limiting the application of this assay to screening small lists of candidate drugs. A larger screen of untargeted drug libraries would not be feasible using an IHC approach. Future developments in this protocol to facilitate larger drug screens would require the integration of a more readily quantifiable cell death marker. While these are available, the irregularity of retinal organoids in size, presence or absence of non-retinal tissue appendages, and variability in photoreceptor layer quality makes it difficult to normalize results across organoids. We expect that future developments to increase the throughput of human organoid disease models to screen large drug libraries will vastly improve our ability to discover, modify, and validate drugs that will advance from preclinical trials to approved therapeutics.

Modeling human disease in cell culture and animal models has provided invaluable tools for the discovery, modification, and validation of pharmacologic therapeutics, allowing them to advance from candidate drug to approved therapy. Although a combination of in vitro and non-human in vivo models has long been a critical component of the drug development pipeline they frequently fail to predict the clinical performance of novel drug candidates1. There is a clear need for the development of technologies that bridge the gap between simplistic human cellular monocultures and clinical trials. Recent technological advances in self-organized three-dimensional tissue cultures, organoids, have improved their fidelity to the tissues they model making them promising tools in the preclinical drug development pipeline2.
A major advantage of human cell culture over non-human in vivo models is the ability to replicate the specific intricacies of human metabolism which can vary considerably even between higher order vertebrates such as humans and mice3. However, this specificity can be overshadowed by a loss in tissue complexity; such is the case for retinal tissue where multiple cell types are intricately interwoven and have a unique symbiotic metabolic interplay between cellular subtypes that cannot be replicated in a monoculture4. Human organoids, which provide a facsimile of complex human tissues with the accessibility and scalability of cell culture, have the potential to overcome the deficiencies of these disease modeling platforms.
Retinal organoids derived from stem cells have proven to be particularly faithful in modeling the complex tissue of the human neural retina5. This has made the retinal organoid model a promising technology for the study and treatment of retinal disease6,7. To date much of the disease modeling in retinal organoids has focused on monogenic retinal diseases where retinal organoids are derived from iPSC lines with disease-causing genetic variants7. These are generally highly penetrant mutations that manifest as developmental phenotypes. Less work has been effectively done on aging diseases where genetic mutations and environmental stressors impact tissue that has developed normally. Neurodegenerative diseases of aging can have complex genetic inheritance and contributions from environmental stressors that are inherently difficult to model using short-term cell cultures. However, in many cases these complex diseases can coalesce on common cellular or metabolic stressors that, when tested on a fully developed human tissue, can provide powerful insights into neurodegenerative diseases of aging8.
The late-onset macular degenerative disease, macular telangiectasia type II (MacTel), is a great example of a genetically complex neurodegenerative disease that coalesces on a common metabolic defect. MacTel is an uncommon retinal degenerative disease of aging that results in photoreceptor and M&#252;ller glia loss in the macula, leading to a progressive loss in central vision9,10,11,12,13. In MacTel, an undetermined, possibly multifactorial, genetic inheritance drives a common reduction in circulating serine in patients, resulting in an increase in a neurotoxic lipid species called deoxysphingolipids (deoxySL)14,15. To prove that accumulation of deoxySL is toxic to the retina and to validate potential pharmaceutical therapeutics, we developed this protocol to assay photoreceptor toxicity in human retinal organoids14.
Here we outline a specific protocol for differentiating human retinal organoids, establishing a toxicity and rescue assay using organoids, and quantifying outcomes. We provide a successful example where we determine the tissue-specific toxicity of a suspected disease-causing agent, deoxySL, and validate the use of a safe generic drug, fenofibrate, for the potential treatment of deoxySL-induced retinal toxicity. Previous work has shown that fenofibrate can increase the degradation of deoxySL and lower circulating deoxySL in patients, however, its efficacy in reducing deoxySL-induced retinal toxicity has not been tested16,17. Although we present a specific example, this protocol can be utilized to evaluate the effect of any number of metabolic/environmental stressors and potential therapeutic drugs on retinal tissue.

<table><tbody><tr><td>0.5M EDTA</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>125mL Erlenmeyer Flasks</td><td>VWR</td><td>89095-258</td><td></td></tr><tr><td>1-deoxysphinganine</td><td>Avanti</td><td>860493</td><td></td></tr><tr><td>B27 Supplement, minus vitamin A</td><td>Gibco</td><td>12587010</td><td></td></tr><tr><td>Beaver 6900 Mini-Blade</td><td>Beaver-Visitec</td><td>BEAVER6900</td><td></td></tr><tr><td>D-(+)-Sucrose</td><td>VWR</td><td>97061-432</td><td></td></tr><tr><td>DAPI</td><td>Thermo-fisher</td><td>D1306</td><td></td></tr><tr><td>Dispase II, powder</td><td>Gibco</td><td>17105041</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Gibco</td><td>11995073</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11330</td><td></td></tr><tr><td>Donkey anti-rabbit Ig-G, Alexa Fluor plus 555</td><td>Thermo-fisher</td><td>A32794</td><td></td></tr><tr><td>donkey serum</td><td>Sigma</td><td>D9663-10ML</td><td></td></tr><tr><td>FBS, Heat Inactivated</td><td>Corning</td><td>45001-108</td><td></td></tr><tr><td>Fenofibrate</td><td>Sigma</td><td>F6020</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Heparin</td><td>Stemcell Technologies</td><td>7980</td><td></td></tr><tr><td>In Situ Cell Death Detection Kit, Fluorescin</td><td>Sigma</td><td>11684795910</td><td></td></tr><tr><td>Matrigel, growth factor reduced</td><td>Corning</td><td>356230</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>mTeSR 1</td><td>Stemcell Technologies</td><td>85850</td><td></td></tr><tr><td>N2 Supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Pierce 16% Formaldehyde</td><td>Thermo-fisher</td><td>28906</td><td></td></tr><tr><td>Rabbit anti-Recoverin antibody</td><td>Millipore</td><td>AB5585</td><td></td></tr><tr><td>Sodium Citrate</td><td>Sigma</td><td>W302600</td><td></td></tr><tr><td>Steriflip Sterile Disposable Vacuum Filter Units</td><td>MilliporeSigma</td><td>SE1M179M6</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T0625</td><td></td></tr><tr><td>Tissue Plus- O.C.T. compound</td><td>Fisher Scientific</td><td>23-730-571</td><td></td></tr><tr><td>Tissue-Tek Cryomold</td><td>EMS</td><td>62534-10</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Ultra-Low Attachment 6 well Plates</td><td>Corning</td><td>29443-030</td><td></td></tr><tr><td>Ultra-Low Attachment 75cm2 U-Flask</td><td>Corning</td><td>3814</td><td></td></tr><tr><td>Vacuum Filtration System</td><td>VWR</td><td>10040-436</td><td></td></tr><tr><td>Vectashield-mounting medium</td><td>vector Labs</td><td>H-1000</td><td></td></tr><tr><td>wax pen-ImmEdge</td><td>vector Labs</td><td>H-4000</td><td></td></tr><tr><td>Y-27632 Dihydrochloride (Rock inhibitor)</td><td>Sigma</td><td>Y0503</td><td></td></tr></tbody></table>

toxicity screens in human retinal organoids for pharmaceutical discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Retinal organoids were generated from a non-MacTel control iPSC line. After organoids reached 26 weeks in culture they were selected and split into experimental groups. Organoids were treated with varying concentrations of deoxySA to determine if deoxySA is toxic to photoreceptors. Four concentrations of deoxySA were tested, from 0 to 1 &#181;M (Figure 2) and organoids were treated for 8 days, with media changes every other day. Cell death in response to deoxySA is concentration-dependent and detectible in as little as 50 nM deoxySA (Figure 2D). The highest concentration tested, 1 &#181;M deoxySA, gave a robust effect while maintaining the integrity of the retinal organoid (Figure 2B,D). A higher concentration of 5 &#181;M deoxySA caused disintegration of organoids within a few days (data not shown). The results of the deoxySA dose response determined the optimal concentration of deoxySA for future toxicity experiments. The 1 &#181;M deoxySA dose resulted in a substantial cell death without oversaturating toxicity, as was observed at 5 &#181;M.
To test the ability of fenofibrate to rescue deoxySA-induced cell death, retinal organoids were treated with either 1 &#181;M deoxySA, 1 &#181;M deoxySA plus 20 &#181;M fenofibrate, or a no treatment vehicle control (Figure 2A-C,E). The 20 &#181;M fenofibrate treatment prevented deoxySA-induced toxicity in the photoreceptors of retinal organoids, significantly reducing cell death by approximately 80% after 4 days of treatment (Figure 2A-C,E)16. Additional lipid-altering drugs were tested using the same protocol, including fumonosin-B1, which showed a complete rescue of deoxySA toxicity. Related lipid species were also tested to identify the specific downstream sphingolipid metabolite that leads to photoreceptor cell death14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62269/62269fig1v2.jpg" /> 
Figure 1: Representative bright field images of organoids under an inverted light microscope. (A) 4-week-old organoids at 2 days following detachment show appropriate retinal layering (white *) or non-retinal organoid development. (B) Developing organoids at week 13. (C) Retinal organoids at week 28 with clear retinal layering and well-formed outer segments projecting from the outer photoreceptor layer (white bar). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62269/62269fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62269/62269fig2v2.jpg" /> 
Figure 2:&#160;Representative confocal images of cell death. Representative confocal images of cell death (TUNEL, green) within the photoreceptor layer (Recoverin, red) of the retinal organoid following 4 days of treatment with either control media (A), 1 &#181;M deoxySA (B), or 1 &#181;M&#160;deoxySA with 20 &#181;M fenofibrate (C). (D) Quantification of cell death in human photoreceptors following treatment of retinal organoid tissue with varying concentrations of deoxySA. (E) Quantification of cell death in human photoreceptors following treatment of retinal organoid tissue with control media (n=6), 1 &#181;M deoxySA (n=22), 1 &#181;M deoxySA + 20 &#181;M fenofibrate (n=21). *p&#60;0.05, ***p&#60;0.001 with one way ANOVA and post hoc Tukey test. Data derived from New England Journal of Medicine, Gantner, M., Eade, K., and Wallace. M., et al. Serine and lipid metabolism in macular disease and peripheral neuropathy, 381(15), 1422-1433, Copyright &#169; (2019) Massachusetts Medical Society. Reprinted with permission.14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62269/62269fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62269/62269fig3v3.jpg" /> 
Figure 3: Screen shots showing a confocal image of a sectioned and stained organoid treated with 1 &#181;M deoxySA viewed using FIJI software. Fluorescent channels have been split in to &#945;-Recoverin (A, red), TUNEL (B, green), and DAPI (C, blue). (A) The photoreceptor area has been outlined using the polygon tool selected in the toolbar (top left). (B,C) Cell counts using the multi-point tool, selected in the tool bar, of TUNEL positive (B, green) and DAPI positive (C, blue) cells within the photoreceptor layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62269/62269fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery at JoVE.com

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

toxicity-screens-human-retinal-organoids-for-pharmaceutical

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.2K Views.  Lowy Medical Research Institute. Dit protocol biedt een preklinische benadering voor het ontdekken en valideren van farmaceutische behandelingen voor de metabole retinale ziekten direct in menselijk netvliesweefsel. Deze techniek combineert de complexiteit van het metabolisme in menselijke weefsels met de veelzijdigheid en doorvoer van celkweek. We hebben deze test gebruikt om preklinische gegevens te verkrijgen voor de behandeling van de retinale degeneratieve ziekte, MacTel, met behulp van een gemeenschappelijk en veilig l...

Video: Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

Cancer Research

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Developmental Biology

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

Generating Neural Retina from Human Pluripotent Stem Cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

Author Spotlight: Simple and Efficient Neural Retina Organoid Production for Disease Modeling

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Neuroscience

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Medicine

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

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