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.
