generation of human retinal organoids and co-culturing with microglia for disease modeling and drug screening

Microglia-Integrated Retinal Organoids: A Model for Studying Retinal Diseases

As the limited source of the human retina, the differentiation of human stem cells into three-dimensional (3D) retinal organoids represents a promising in vitro model for simulating the retina1. It contains different cell types in the retina, including photoreceptors, retinal ganglion cells, bipolar cells, M&#252;ller cells, horizontal cells, and astrocytes2. This model enables the emulation and study of both retinal development mechanisms and the pathogenesis of retinal diseases. However, due to the directional differentiation method, retinal organoids were derived from the neuroectoderm3, lacking many other cell types originating from different germ layers, such as microglia from the yolk sac and perivascular cells from the mesoderm4,5,6.
At present, many retinal diseases, such as retinitis pigmentosa7, glaucoma8, and retinoblastoma9, have been proven to be closely related to microglia within the retina. However, due to the lack of proper research models, specific mechanisms illustrating the relationship between microglia and these diseases still remain unclear. While mice have served as a favorable model for studying retinal diseases, recent studies have highlighted significant differences between mouse and human microglia in terms of lifespan, proliferation rate, and the absence of human homologous genes10,11. These findings suggested that conclusions drawn from mouse models may not be entirely reliable, emphasizing the importance of constructing human retinal organoids containing microglia.
Over the past few decades, various methods for the 3D differentiation of retinal organoids have been developed12,13. To facilitate the co-culture operation of microglia within retinal organoids, we have selected a differentiation method involving a transition from adherent to suspension culture. This approach successfully enables microglia to be incorporated into the retinal organoids, maintaining them for at least 60 days14.

Generation of Human Microglia to Combine Them with Retinal Organoids for Improved Disease Modeling

Assembling Retinal Organoids with Microglia

To begin, obtain the human embryonic stem cells in the stem cell medium with a cell density of 80 to 90%After removing the spent medium, rinse the cells in each well with one milliliter of 1X DPBS. Incubate the stem cell colonies in each well with one milliliter of 0.5 millimolar EDTA for approximately five minutes at 37 degrees Celsius. Check the colony morphology to confirm that the edges are slightly curled up with loose gaps.Aspirate the EDTA, and if necessary, incubate cells at 37 degrees Celsius for another one to two minutes, but not exceeding eight minutes. Gently rinse the cells in each well with six milliliters of medium A containing six microliters of 10 millimolar ROCK inhibitor Y-27632 solution. Gently tap the bottom of the dish to help rinse the cells.After 24 hours, observe the cells under the microscope to confirm the embryo body formation. Using a 10 milliliter pipette, collect the embryo bodies into a 15 milliliter centrifugal tube and wait for the embryo bodies to settle to the bottom for five minutes. Then carefully aspirate the supernatant and resuspend the embryo bodies in six milliliters of fresh medium A.Transfer them into a new low adhesion well of a six-well plate and culture in a 37 degrees Celsius incubator.On day four, coat a 10 centimeter dish with three milliliters of 0.1%fish gelatin solution and incubate for at least one hour in a 5%carbon dioxide, 37 degrees Celsius incubator. Then remove the gelatin solution and rinse the dish once with five to six milliliters of 1X DPBS. Carefully collect the embryo bodies into a 15 milliliter centrifugal tube and wait for the embryo bodies to settle to the bottom In five minutes.Gently resuspend the embryo bodies with 15 milliliters of medium B and transfer them to the coated 10 centimeter dish. Culture in a 37 degrees Celsius, 5%carbon dioxide incubator for one week after 49 days. Centrifuge the cells at 200 x g for five minutes at room temperature.Carefully aspirate the supernatant and resuspend the cells with two milliliters of fresh medium C.Transfer into a new low adhesion well of a six-well plate. Incubate the plate in a 5%carbon dioxide, 37 degrees Celsius incubator. On day 56, replace the spent medium with two milliliters of medium C per well.Examine the plate under a microscope to identify the branched microglia adhere to the bottom of the low adhesion well. To harvest microglia for co-culturing, gently replace the medium C in each well with one milliliter Accutase and incubate at 37 degrees Celsius for three minutes. Using a five milliliter pipette, collect the microglia cells into a 15 milliliter tube and centrifuge the cells at 200 x g for five minutes at room temperature.After discarding the supernatant, suspend the microglia with one milliliter of medium E.

Generation of Human Retinal Organoids and Co-Culturing with Microglia for Disease Modeling and Drug Screening

To begin, obtain human microglia from the human embryonic stem cells and harvest them on day 56. For retinal organoid derivation, culture the human embryonic stem cells in a stem cell medium until the cell density reaches 80 to 90%Add one milliliter dispase per well to the culture cells and incubate at 37 degrees Celsius for five minutes. After removing dispase, add one milliliter of medium D to each well.Cut the cells into small pieces. With a 10 microliter pipette, gently collect all the cell pieces and medium into a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 200 x g for five minutes.After removing the supernatant, gently resuspend the cells in 200 microliters of a cold matrix. Move the 1.5 milliliter microcentrifuge tube into an incubator for 20 minutes. After 20 minutes in the incubator, the matrix will solidify.Prepare a 10 centimeter dish with 15 milliliters of medium D.Resuspend the matrix with medium D and shake the Petri dish gently. Place the dish in an incubator for five days. On day 12, replace the medium with three milliliters of dispase.After five minutes, aspirate the dispase and add 15 milliliters of medium E.Introduce the harvested microglia to digested retinal organoids on day 12. On day 19, gently swirl the plate to aggregate the organoids to the center of the dish and replace the medium E with medium F.Finally, transfer the organoids with medium F to a new suspension dish. By day 30, retinal organoids co-cultured with microglia showed significant GFP autofluorescence, indicating the presence of microglia.The co-cultured retinal organoids displayed clear immunofluorescence signals for the photoreceptor marker CRX and the microglial marker IBA1.

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Research

JoVE Journal

Neuroscience

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal the mechanism of retinal development and the occurrence of retinal disease. Microglia (MG) are unique resident macrophages in the retina and central nervous system (CNS), serving crucial immunity functions. However, retinal organoids lack microglia since their differentiation origin is the yolk sac. The specific pathogenesis of microglia in these retinal diseases remains unclear; therefore, the establishment of a microglia-incorporated retinal organoid model turns out to be necessary. Here, we successfully constructed a co-cultured model of retinal organoids with microglia derived from human stem cells. In this article, we differentiated microglia and then co-cultured to retinal organoids in the early stage. As the incorporation of immune cells, this model provides an optimized platform for retinal disease modeling and drug screening to facilitate in-depth research on the pathogenesis and treatment of retinal and CNS-related diseases.

Microglia are unique resident immune cells in the retina, playing crucial roles in various retinal degenerative diseases. Generating a co-culture model of retinal organoids with microglia can facilitate a better understanding of the pathogenesis and development progress of retinal diseases.

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal ...