Name: toxicity screens in human retinal organoids for pharmaceutical discovery
Uploaded: 2021-03-04T15:00:00
Description: Watch this Scientific Journal Video about Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery at JoVE.com

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.

1. Thawing, passaging, and expanding iPSCs/ESCs
NOTE: For all cell culturing steps, use best practices to maintain a sterile cell culture.
<ol>
	<li>Coat a 6-well cell culture plate with basement membrane matrix medium.
	<ol>
		<li>To prepare 1x of this medium, follow product specifications or dilute 75 &#181;L cold matrix medium with 9 mL of DMEM/F12. Add 1.5 mL of freshly prepared 1x medium per well in a 6-well plate. Incubate at 37 &#176;C for 30 min.</li>
		<li>Aspirate off the basement ...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+tissues+and+strategies+to+overcome+challenges+in+drug+development.">Engineered tissues and strategies to overcome challenges in drug development.</a> Advances in Drug Delivery Reviews. 158, 116-139 (2020).</li><li>Demetrius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+men.+When+it+comes+to+studying+ageing+and+the+means+to+slow+it+down,+mice+are+not+just+small+humans.">Of mice and men. When it comes to studying ageing and the means to slow it down, mice are not just small humans.</a> EMBO Reports. , 39-44 (2005).</li><li>Lindsay, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyruvate+kinase+and+aspartate-glutamate+carrier+distributions+reveal+key+metabolic+links+between+neurons+and+glia+in+retina.">Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina.</a> Proceedings of the National Academy of Sciences U. S. A. 111 (43), 15579-15584 (2014).</li><li>Cowan, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+types+of+the+human+retina+and+its+organoids+at+single-cell+resolution.">Cell types of the human retina and its organoids at single-cell resolution.</a> Cell. 182 (6), 1623-1640 (2020).</li><li>Kruczek, K., Swaroop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-derived+retinal+organoids+for+disease+modeling+and+development+of+therapies.">Pluripotent stem cell-derived retinal organoids for disease modeling and development of therapies.</a> Stem Cells. 38 (10), 1206-1215 (2020).</li><li>Sinha, D., Phillips, J., Phillips, M. J., Gamm, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+retinal+development+and+disease+with+human+pluripotent+stem+cells.">Mimicking retinal development and disease with human pluripotent stem cells.</a> Investigative Ophthalmology and Visual Science. 57 (5), 1-9 (2016).</li><li>Gan, L., Cookson, M. R., Petrucelli, L., La Spada, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Converging+pathways+in+neurodegeneration,+from+genetics+to+mechanisms.">Converging pathways in neurodegeneration, from genetics to mechanisms.</a> Nature Neuroscience. 21 (10), 1300-1309 (2018).</li><li>Aung, K. Z., Wickremasinghe, S. S., Makeyeva, G., Robman, L., Guymer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+estimates+of+macular+telangiectasia+type+2:+the+Melbourne+collaborative+cohort+study.">The prevalence estimates of macular telangiectasia type 2: the Melbourne collaborative cohort study.</a> Retina. 30 (3), 473-478 (2010).</li><li>Chew, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ciliary+neurotrophic+factor+on+retinal+neurodegeneration+in+patients+with+macular+telangiectasia+type+2:+A+randomized+clinical+trial.">Effect of ciliary neurotrophic factor on retinal neurodegeneration in patients with macular telangiectasia type 2: A randomized clinical trial.</a> Ophthalmology. 126 (4), 540-549 (2019).</li><li>Gass, J. D., Blodi, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+juxtafoveolar+retinal+telangiectasis.+Update+of+classification+and+follow-up+study.">Idiopathic juxtafoveolar retinal telangiectasis. Update of classification and follow-up study.</a> Ophthalmology. 100 (10), 1536-1546 (1993).</li><li>Klein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+macular+telangiectasia+type+2+in+the+Beaver+Dam+eye+study.">The prevalence of macular telangiectasia type 2 in the Beaver Dam eye study.</a> American Journal of Ophthalmology. 150 (1), 55-62 (2010).</li><li>Powner, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Muller's+cells+and+photoreceptors+in+macular+telangiectasia+type+2.">Loss of Muller's cells and photoreceptors in macular telangiectasia type 2.</a> Ophthalmology. 120 (11), 2344-2352 (2013).</li><li>Gantner, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine+and+lipid+metabolism+in+macular+disease+and+peripheral+neuropathy.">Serine and lipid metabolism in macular disease and peripheral neuropathy.</a> New England Journal of Medicine. 381 (15), 1422-1433 (2019).</li><li>Scerri, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analyses+identify+common+variants+associated+with+macular+telangiectasia+type+2.">Genome-wide analyses identify common variants associated with macular telangiectasia type 2.</a> Nature Genetics. 49 (4), 559-567 (2017).</li><li>Alecu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+1-deoxysphingolipids+are+metabolized+by+a+cytochrome+P450-dependent+pathway.">Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway.</a> Journal of Lipid Research. 58 (1), 60-71 (2017).</li><li>Othman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fenofibrate+lowers+atypical+sphingolipids+in+plasma+of+dyslipidemic+patients:+A+novel+approach+for+treating+diabetic+neuropathy.">Fenofibrate lowers atypical sphingolipids in plasma of dyslipidemic patients: A novel approach for treating diabetic neuropathy.</a> Journal of Clinical Lipidology. 9 (4), 568-575 (2015).</li><li>Ohlemacher, S. K., Iglesias, C. L., Sridhar, A., Gamm, D. M., Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+highly+enriched+populations+of+optic+vesicle-like+retinal+cells+from+human+pluripotent+stem+cells.">Generation of highly enriched populations of optic vesicle-like retinal cells from human pluripotent stem cells.</a> Curr Protoc Stem Cell Biol. 32, 1-20 (2015).</li><li>Zhong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+three-dimensional+retinal+tissue+with+functional+photoreceptors+from+human+iPSCs.">Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.</a> Nature Communication. 5, 4047 (2014).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472 (7341), 51-56 (2011).</li><li>Wahlin, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+outer+segment-like+structures+in+long-term+3D+retinas+from+human+pluripotent+stem+cells.">Photoreceptor outer segment-like structures in long-term 3D retinas from human pluripotent stem cells.</a> Science Reports. 7 (1), 766 (2017).</li><li>Capowski, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+and+staging+of+3D+human+retinal+organoids+across+multiple+pluripotent+stem+cell+lines.">Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.</a> Development. 146 (1), (2019).</li><li>Luo, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+system+for+effective+derivation+of+three-dimensional+retinal+tissue+via+Wnt+signaling+regulation.">An optimized system for effective derivation of three-dimensional retinal tissue via Wnt signaling regulation.</a> Stem Cells. 36 (11), 1709-1722 (2018).</li><li>Ovando-Roche, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bioreactors+for+culturing+human+retinal+organoids+improves+photoreceptor+yields.">Use of bioreactors for culturing human retinal organoids improves photoreceptor yields.</a> Stem Cell Research Therapy. 9 (1), 156 (2018).</li></ol>

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 p...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 an...

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 ...

This protocol provides a preclinical approach to discovering and validating pharmaceutical treatments for the metabolic retinal diseases directly within human retinal tissue. This technique combines the complexity of metabolism in human tissues with the versatility and throughput of cell culture. We have used this assay to acquire preclinical data for treatment of the retinal degenerative disease, MacTel, using a common and safe lipid altering drug, fenofibrate, which is now in clinical safety trials.This approach can be applied to any number of common retinal stressors that lead to disease including nutrient deficiency, hypoxia, light toxicity, and other toxic lipids. At week 24 of culture, check the organoids for the formation of rudimentary segments on the outside of the organoid. By week 26 to 28, the outer segments should have formed a thick layer.When a thickened doubter segment can be observed, dilute one millimolar deoxySA stock solution to 0.51. One and two micromolar concentrations in three milliliters of retinal differentiation medium plus. Using a dissection microscope and a sterile probe, select four groups of organoids of approximately the same size and shape with a minimum of five organoids per group, and split the groups into individual wells of an ultra-low attachment 6-well plate.When all of the organoids have been collected, remove as much medium from each well as possible and add one concentration of the deoxySA solution to each well. Add the vehicle control to the fourth group of organoids. After two days of culture, replace the culture supernatants with the appropriate fresh deoxySA or control dilution.After four days of culture, embed cryo section and stain the organoids according to standard protocols to allow assessment of the cell death within each culture. After staining, use a five to 10x magnification to locate a region of the sliced organoid section that is intact, well formed, and in a plane that samples a representative cross section of the organoid. The section should have a distinct outer nuclear layer of photoreceptors that is at least a few cells thick, and a separate and distinct layer of nuclei that do not have recover and staining.Frame the image and increase the magnification to 20x. Then, frame the slide again to fill the image with as much of the photoreceptor layer as possible. Set the Z-plane and set the upper end lower limits of the Z-range to image the entire depth of the slice.Then image all three channels of fluorophore in a Z-stack acquisition, and save the image in a file format that preserves the ratio of area per pixel. To quantify the number of dead cells in each sample, open the image stacks in Fiji. In the bio format's import options window, confirm that no boxes under the split into separate windows section are checked and select Image, Stacks, and Z project to create a new image for quantification.Select the image channel that shows the recover and staining and use the polygon selection tool to outline a continuous region of photoreceptors in the image. Click Analyze and Set Measurements and select the Area box. Click Analyze and Measure, to measure the area of the photoreceptors, to count the dying cells.The measurement will appear in a pop-up window. To count the apoptosis positive nuclei in the selected photoreceptor region, right click on the point tool to select the multipoint tool and select the apoptosis positive staining that overlaps with a DAPI positive nuclei and recover and staining. Toggle between the channels to validate the positive cells.Then divide the number of apoptosis positive cells by the area to obtain a normalized value for the cell death within the photoreceptors per organoid. After determining the deoxySA concentration that induces the target cell death, use a sterile probe to select three groups of organoids of approximately the same size and shape with a minimum of five organoids per group. And add the groups to individual wells of an ultra-low attachment 6-well plate.Add the target cell death concentration of deoxySA, the target cell death concentration of deoxySA supplemented with the drug of interest or the control medium to the appropriate well or organoids. After two days, replace the supernatant in each well with the appropriate concentration of fresh treatment or control medium. After four days of culture, assess the cultures for cell death as demonstrated.In this representative analysis, retinal organoids generated from a non macular telangiectasia type 2 control induced pluripotent stem cell line were treated with four concentrations of deoxySA. Cell death in response to deoxySA was concentration dependent and detectable in as little as 50 nanomolar deoxySA. The highest concentration tested induced a robust cell death while still maintaining the integrity of the retinal organoid.Treatment with 20 micromolar fenofibrate prevented deoxySA induced toxicity in the photo receptors of retinal organoids, significantly reducing cell death by approximately 80%after four days of treatment. Remember to select well formed and healthy organoids to ensure that the assay is being performed in retinal cells. The organoids can be processed for RNA collection to quantify gene expression, allowing the evaluation of stress signals and the validation of toxic effects and rescues.Using this technique, we have been able to assay the downstream metabolism of multiple different lipids in organoids, opening up the study of lipid metabolism in human retinas.

Research

JoVE Journal

Bioengineering

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...