This study is supported by the National Natural Science Foundation of China (82101145) and the Beijing Natural Science Foundation (Z200014).

This study was approved by the Institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. HESCs cell line H9 was from the WiCell Research Institute. Pre-warm the cell culture medium at room temperature (RT) for 30 min before the experiment.
1. Generation of human microglia
<ol>
	<li>Culture the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 106 cells in each well.</li>
	<li>Aspirate the stem cell medium and rinse the cells with 1x DPBS (1 mL/well of 6 well plate). The next step requires 2 x 107-1 x 108 cells. If not enough, combine cells of 2-5 wells to do the following steps.</li>
	<li>Dissociate the stem cell colonies using the 0.5 mM EDTA solution for about 5 min in a 37 &#176;C incubator (1 mL/well of 6 well plate).</li>
	<li>Check colony morphology after 5 min. When the colony edges are slightly curled up with loose gaps, cells are ready to be differentiated and aspirate the EDTA. If not, incubate cells at 37 &#176;C for another 1-2 min. Do not incubate for longer than 8 min.</li>
	<li>Gently rinse the cells in each well using 6 mL of Medium A (Table 1) with 6 &#181;L of 10 mM ROCK inhibitor Y-27632 solution. Gently tap the bottom of the dish to help rinse the cells. Set the day as day 0. 
	NOTE: Do not pipette the cells. Cells will form embryoid bodies (EBs) the next day.</li>
	<li>After 24 h, observe the cells under the microscope to confirm the EB formation. Collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.</li>
	<li>Carefully aspirate the supernatant and resuspend the EBs with 6 mL of fresh Medium A and transfer them into a new low-adhesion well of 6-well plate. Culture in a 37 &#176;C incubator. 
	NOTE: The purpose of the well transferring on day 1 is to reduce the influence of a large number of dead cells during the process of EB formation, which may affect the state of cell growth.</li>
	<li>Refresh the fresh Medium A daily until day 4 (6 mL/well of 6-well plate).</li>
	<li>On day 4, coat a 10 cm dish with 3 mL of 0.1% fish gelatin solution for at least 1 h in a 5% CO2, 37 &#176;C incubator. 
	NOTE: If the coating time is less than 1 h, it is difficult to carry out the following steps.</li>
	<li>Carefully collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.</li>
	<li>Remove the 0.1% gelatin solution and rinse the 10 cm dish once with 5-6 mL of 1x DPBS. Remove the DPBS.</li>
	<li>Gently resuspend the EBs with 15 mL of Medium B (Table 1) in the 15 mL centrifugal tube and transfer them to the coated 10 cm dish. Culture in a 37 &#176;C, 5% CO2 incubator for 1 week. 
	NOTE: In the first 7 days, do not move the dish.</li>
	<li>Refresh with fresh Medium B once a week until day 49 (15 mL/10 cm dish), and examine the cells under the microscope once a week. 
	NOTE: After day 49, several secretory cells can be found in the supernatant in the culture dish.</li>
	<li>Centrifuge the supernatant cells at 200 x g for 5 min at RT.</li>
	<li>Carefully aspirate the supernatant and resuspend the cells with 2 mL of fresh Medium B and transfer into a new low-adhesion well of 6-well plate. Incubate in a 5% CO2, 37 &#176;C incubator. 
	NOTE: In the next 7 days, do not move the plate. The microglia could adhere to the bottom of the low-adhesion well.</li>
	<li>On day 56, replace with Medium C (Table 1) (2 mL/well of 6-well plate). The microglia adhere to the bottom of the low-adhesion well and will look branched under the microscope. 
	NOTE: By changing Medium C every 3 days (2 mL/well of 6-well plate), the microglia could be cultivated in Medium C for about 15 days.</li>
</ol>
2. Generation of human ROs and co-culture the ROs with microglia
<ol>
	<li>Cultured the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 106 cells in each well.</li>
	<li>Add Dispase (1 mL/well of 6 well plate) to the culture cell. Incubate at 37 &#176;C for 5 min. Aspirate the Dispase solution from the well.</li>
	<li>Add Medium D (Table 1) (1 mL/well of 6 well plate) to the well. Cut the cell into small pieces with a 10 &#181;L pipette.</li>
	<li>Gently collect all the cell pieces and medium in a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge at 200 x g for 5 min and remove the supernatant.</li>
	<li>Gently resuspend the cells in 200 &#181;L of a cold matrix. Move the 1.5 microcentrifuge tube into an incubator for 20 min. Operate fast because the matrix will solidify at room temperature (RT). After 20 min in the incubator, the matrix will solidify.</li>
	<li>Prepare a 10 cm dish with 15 mL Medium D. Resuspend the Matrix with Medium D and shake the Petri dish gently.</li>
	<li>Then, put the dish in an incubator for 5 days. Do not move the plate. Set the day as day 0 (ROs). The cells will adhere in 3 days.</li>
	<li>On day 5 (ROs) and day 10 (ROs), refresh the medium with Medium D.</li>
	<li>On day 12 (ROs), remove the medium and add 3 mL of Dispase to each dish for 5 min.</li>
	<li>Aspirate the Dispase and add 15 mL of Medium E (Table 1).</li>
	<li>Harvest microglia: On day 56 (MGs), gently aspirate the Medium C, add Accutase (1 mL/well of a 6-well plate), and incubate at 37 &#176;C for 3 min.</li>
	<li>Collect the microglia cells into a 15 mL centrifugal tube with a 5 mL pipet. Centrifuge cells at 200 x g at RT for 5 min.</li>
	<li>Gently aspirate the supernatant and suspend the microglia with 1 mL of Medium E. Add microglia to digested ROs on day 12. 
	NOTE: On day 12 (ROs), the cells are adhesive. On day 13 (ROs), the cells are found suspending in Medium E. If the medium changes yellow from day 12 (ROs) to day 19 (ROs), refresh the medium with Medium E.</li>
	<li>On day 19 (ROs), aggregate the organoids to the center of the dish by gently swirling the plate. Gently aspirate Medium E and refresh the medium with Medium F.</li>
	<li>Transfer the organoids with Medium F to a new suspension dish.</li>
	<li>Change the medium once a week and select organoids for experiments.</li>
</ol>

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

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(170), e62298 (2021).</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>Ginhoux, F., 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=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Kierdorf, K., 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=Microglia+emerge+from+erythromyeloid+precursors+via+pu.1-+and+irf8-dependent+pathways.">Microglia emerge from erythromyeloid precursors via pu.1- and irf8-dependent pathways.</a> Nat Neurosci. 16 (3), 273-280 (2013).</li><li>Schulz, C., 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=A+lineage+of+myeloid+cells+independent+of+myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>O'koren, E. G., 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=Microglial+function+is+distinct+in+different+anatomical+locations+during+retinal+homeostasis+and+degeneration.">Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration.</a> Immunity. 50 (3), 723-737.e7 (2019).</li><li>Margeta, M. 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=Apolipoprotein+E4+impairs+the+response+of+neurodegenerative+retinal+microglia+and+prevents+neuronal+loss+in+glaucoma.">Apolipoprotein E4 impairs the response of neurodegenerative retinal microglia and prevents neuronal loss in glaucoma.</a> Immunity. 55 (9), 1627-1644.e7 (2022).</li><li>Xu, 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=Enhanced+innate+responses+in+microglia+derived+from+retinoblastoma+patient-specific+IPSCs.">Enhanced innate responses in microglia derived from retinoblastoma patient-specific IPSCs.</a> Glia. 72 (5), 872-884 (2024).</li><li>Gosselin, D., 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+environment-dependent+transcriptional+network+specifies+human+microglia+identity.">An environment-dependent transcriptional network specifies human microglia identity.</a> Science. 356 (6344), eaal3222 (2017).</li><li>Galatro, T. F., 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=Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes.">Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.</a> Nat Neurosci. 20 (8), 1162-1171 (2017).</li><li>Nakano, T., 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-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10 (6), 771-785 (2012).</li><li>Kim, 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=transcriptome+profiling,+and+functional+validation+of+cone-rich+human+retinal+organoids.">transcriptome profiling, and functional validation of cone-rich human retinal organoids.</a> Proc Natl Acad Sci U S A. 116 (22), 10824-10833 (2019).</li><li>Gao, 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=Functional+microglia+derived+from+human+pluripotent+stem+cells+empower+retinal+organ.">Functional microglia derived from human pluripotent stem cells empower retinal organ.</a> Sci China Life Sci. 65 (6), 1057-1071 (2022).</li><li>Zhang, X., Jin, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruct+human+retinoblastoma+in+vitro.">Reconstruct human retinoblastoma in vitro.</a> J Vis Exp. (188), e62629 (2022).</li><li>Park, D. 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=IPS-cell-derived+microglia+promote+brain+organoid+maturation+via+cholesterol+transfer.">IPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer.</a> Nature. 623 (7986), 397-405 (2023).</li><li>Usui-Ouchi, 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=Integrating+human+ipsc-derived+macrophage+progenitors+into+retinal+organoids+to+generate+a+mature+retinal+microglial+niche.">Integrating human ipsc-derived macrophage progenitors into retinal organoids to generate a mature retinal microglial niche.</a> Glia. 71 (10), 2372-2382 (2023).</li><li>Chichagova, V., 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=Incorporating+microglia-like+cells+in+human+induced+pluripotent+stem+cell-derived+retinal+organoids.">Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.</a> J Cell Mol Med. 27 (3), 435-445 (2023).</li></ol>

The authors are not aware of any affiliations, membership, grants, or financial holding that might affect the objectivity of this study.

Due to the restricted availability of the human retina, our current comprehension of retinal inflammatory responses almost comes from animal models. To overcome this limitation, retinal organoids were differentiated. The development of retinal organoid models has been an active area of research, aiming to recapitulate the complexity of the human retina for disease modeling and therapeutic development. Several studies have reported successfully generating retinal organoids from human pluripotent stem cells<sup class="xref...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acctuase</td>
			<td>Stemcell Technologies</td>
			<td>07920</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Thermo</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CRX(M02)</td>
			<td>abnova</td>
			<td>H00001406-M02</td>
			<td>Antibody; dilution as per the manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Anti-IBA1</td>
			<td>Abcam</td>
			<td>ab5076</td>
			<td>Antibody; dilution as per the manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase (1U/mL)</td>
			<td>Stemcell Technologies</td>
			<td>07923</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>C141905005BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biological Industry</td>
			<td>04-002-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G7041-100G</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>H9 cell line</td>
			<td>WiCell Research Institute</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>RD Systems&#160;</td>
			<td>203-IL-050</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-34</td>
			<td>PeproTech</td>
			<td>200-34-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>RD Systems&#160;</td>
			<td>216-MC-500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem cell medium&#160;</td>
			<td>Stemcell Technologies</td>
			<td>5990</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T-8691-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ViVO</td>
			<td>LONZA</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
	</tbody>
</table>

assembling retinal organoids with microglia

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.

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.

The procedure for generating retinal organoids is described in our previous study15. Here, we show the representative results of microglia and co-culture microglia and retinal organoids.
Here, we demonstrate each stage of microglia differentiation (Figure 1A). Day 0 represents the stage of stem cell culture. Then, the stem cells were digested and cultured for EB formation. In the initial 4 days of the process, cells will form EBs (<strong c...

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 ...

assembling-retinal-organoids-with-microglia

Research

JoVE Journal

Neuroscience

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.

716 Views.  Capital Medical University.  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.

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

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