Name: Derivation of a Human Brain Organoid with Microglia Development (Video) | JoVE Journal
Uploaded: 2025-01-17T13:30:00.000+00:00
Duration: 10 min 34 s
Description: Learn to generate human brain organoids containing resident microglia and neurons using iPSC-derived hematopoietic progenitor cells. This method enables study of microglia-neuron interactions during brain development.

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5 minutes

This study is supported by NINDS&#160;intramural research funds.

The original blood samples from healthy adult donors were collected at the Transfusion Medicine Blood Bank of the NIH, and signed informed consent forms were obtained in accordance with the NIH Institutional Review Board.
1. Producing hematopoietic progenitor cells (HPCs) from human iPSCs
NOTE: Human iPSC cells 510 and 507 were used to produce the representative results. The methods of generation and maintenance of the iPSCs can be found in a previous publication16.
<ol>
	<li>Day 0: Coat a 12-well cell plate by adding 500 &#956;L/well of ice-cold Matrigel (basement membrane matrix [BMM]) solution diluted in DMEM/F12 medium and incubate it for at least 30 min at room temperature (RT).</li>
	<li>Remove the coating supernatant completely and replace it with 1 mL of E8 Flex medium per well.</li>
	<li>Check the iPSC culture in a 6 well plate under the microscope to confirm the iPSCs are of high quality and without signs of differentiation. Choose a middle-sized colony by marking it with a marker pen underneath the bottom of the plate.</li>
	<li>Remove the medium from the iPSC culture and add 500 &#956;L of ethylenediaminetetraacetic acid (EDTA) iPSC dissociation buffer (0.5 mM EDTA, 0.45 g/L NaCl in DPBS) to the well.</li>
	<li>Observe under a microscope for signs of cell dissociation when three to four rows of cells from the edges of the colony start to shrink, with empty space appearing between cells. This usually takes 1-3 min.</li>
	<li>Discard the EDTA solution completely. Dislodge the marked iPSC colony by pipetting 1 mL of E8 Flex medium forcefully and directly onto it. Ensure that the colony detaches completely into cell patches containing 20-50 cells each after 1-3 times of pipetting.</li>
	<li>Collect the supernatant containing cell patches and add it to the first well of the coated wells in the 12-well plate.</li>
	<li>Mix the well by pipetting one or two times and transfer 1 mL of the cells into the second well that contains 1 mL of medium.</li>
	<li>Repeat step 1.8 twice to make serial diluted iPSC cultures in a total of four wells.</li>
	<li>Incubate the plate in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>After 24 h, on day 1, check the colonies under the microscope and count the numbers of colonies by going through all the fields in a well continuously. Choose one well that contains 10-20 iPSC colonies.</li>
	<li>Replace the medium with 1 mL of medium A from the hematopoietic Kit. Put the plate back into the incubator for 48 h.</li>
	<li>On day 3, remove 500 &#956;L of spent medium and add 500 &#956;L of fresh medium A.</li>
	<li>On day 4,observe under the microscope and notice significant cell growth and differentiation from the iPSC colonies. Replace the medium with 1 mL of medium B.</li>
	<li>Monitor the cell differentiation under the microscope and do half-medium change with 500 &#956;L of Medium B every other day. The HPC-like cells appear on days 6-7.</li>
	<li>On day 10, observe under the microscope bright single round cells with the morphology of normal HPCs floating in the medium or loosely attached to the single bottom layer of flat cells, with some loose cell aggregates. 
	NOTE: The differentiated HPCs are ready for collection for characterization or further experiment.</li>
	<li>Collect all the differentiated HPCs by pipetting up and down three times using a 1 mL pipette tip to break the cell aggregates and detach the HPCs from the plate surface.</li>
	<li>Add the cells into a 15 mL tube and centrifuge the cells at 300 x g for 5 min. Remove the supernatant and resuspend the cell pellet into 1 mL of medium B.</li>
	<li>Count the cells and adjust the concentration to 1 million cells/mL. At this stage, more than 1 million of HPCs are generated. When checked with flow cytometry, ensure that the purity is more than 85% of cells with CD34+/CD43+. Ensure no significant dead cells (more than 5%) are noticed.</li>
</ol>
2. Developing embryoid bodies from mixed iPSCs and HPCs
<ol>
	<li>On the same day of collecting the HPCs, make sure an iPSC culture also reaches 80% confluence in one well in a 6-well plate (or two wells in a 12-well plate).</li>
	<li>Treat one well of the microwell culture plate&#160;for&#160;EB formation&#160;with 500 &#181;L of anti-adherence rinsing solution by adding it to minimize bubbles.</li>
	<li>Spin the plate at 2000 x g for 5 min in a swinging bucket rotor fitted with plate holders to remove possible bubbles.</li>
	<li>Remove the rinsing solution completely by pipetting and wash the wells twice with 1 mL of DPBS and then 1 mL of DMEM/F12 without generating bubbles.</li>
	<li>Dissociate the iPSCs from the plate by treating the cells with 1 mL of Accutase solution.</li>
	<li>Observe under the microscope until the cells show signs of separation but are still attached to the bottom. This usually happens within 1-3 min of Accutase treatment. Do not over-digest the iPSCs.</li>
	<li>Remove the Accutase solution completely without disturbing the cells and add1 mL of DMEM/F12 medium to the wells. Further, dissociate the cells into single cells by pipetting up and down a couple of times using a 1 mL tip.</li>
	<li>Collect the cells into a 15 mL tube and fill it to 5 mL with DMEM/F12 medium. Spin the cells at 300 x g for 5 min.</li>
	<li>Remove the supernatant by pipetting and resuspend the cells in 1 mL of E8 Flex medium. Count the cells and adjust the cell concentration to 1 million cells per mL in the E8 Flex medium.</li>
	<li>Mix iPSCs with HPCs in a ratio of 2:1 by adding 1 million of iPSCs (1 mL) and a half million of HPCs (500 &#181;L) together. Add all the mixed cells into the previously treated well of the microwell culture plate.</li>
	<li>Add 1 &#181;L of Rock inhibitor Y27632 stock solution (1 mM) per 1 mL medium into the supernatant. Shake the plate from side to side a few times to distribute the cells evenly.</li>
	<li>Spin the microwell culture plate at 300 x g for 5 min in a swinging bucket rotor fitted with plate holders. Observe under the microscope to see if the cells have settled into the microwells. (Supplementary Figure 1A).</li>
	<li>Without disturbing the cells, put the plate in a 37 &#176;C, 5% CO2 cell incubator.</li>
	<li>On day 2, 48 h later, observe the cells under the microscope for the formation of EBs. When the EBs are clearly formed, continue to the next step (Supplementary Figure 1B).</li>
	<li>Treat a 24-well cell culture plate with anti-adherence rinsing buffer (500 &#181;L) for 15 min to make a low attachment plate.</li>
	<li>Remove the rinsing buffer completely and wash the wells twice with 1 mL of DPBS, then add 1 mL of E8 Flex medium into the wells.</li>
	<li>Resuspend the EBs in the microwell culture plate by pipetting them with a 1 mL wide orifice pipette tip for a few times.</li>
	<li>For each well of the low adherence treated 24 well plates, collect and add 100 &#181;L of medium containing EBs. This will result in 10-20 EBs per well. Add E8 Flex medium to the old well containing the leftover EBs as backups.</li>
	<li>Incubate the plate in a 37 &#176;C, 5% CO2 cell incubator for 48 h.</li>
</ol>
3. 3D neural organoid induction, proliferation, and maturation
<ol>
	<li>On day 2 after EB formation, thaw aliquots of BMM on ice for at least 30 min.</li>
	<li>Use a pre-chilled 20 &#181;L tip, and add 15 &#181;L ice-cold BMM drop wisely on top of the medium to coat the EBs. This makes a film of BMM that EBs will attach and grow onto.</li>
	<li>Put the plate in a 37 &#176;C, 5% CO2 cell incubator.</li>
	<li>On day 4, after the EB formation, remove 500 &#181;L of medium carefully without discarding EBs and repeat BMM coating by following step 3.2.</li>
	<li>Immediately start neural induction by adding 500 &#181;L of PSC Neural Induction Medium on top of cells.</li>
	<li>On days 6 and 8, perform half medium change by carefully removing 500 &#181;L of medium without touching cells and adding 500 &#181;L of neural induction medium.</li>
	<li>On days 10, 12, and 14, perform half medium change with neural stem cell (NSC) medium: Knockout DMEM/F12 + 1x Glutamax supplement + 1x Neural supplement + bFGF + EGF.</li>
	<li>On day 15, transfer the whole well of spheres with medium to a new 12-well plate treated with anti-adherence rinsing solution as previously described. Add 500 &#181;L of neuronal maturation medium (DMEM/F12 + 1x N2 supplement + 1x B27 supplement + 1x antibiotics-antimycotic) on top of the culture.</li>
	<li>Perform half-medium change with neuronal maturation medium every other day.</li>
	<li>During the stage of maturation, if there are signs of nutrition depletion, such as color change of medium, move the organoid to 6 well plates, which hold up to 3 mL of medium per well.</li>
	<li>Optionally, 17 days after the EB formation, supplement the maturation medium with cytokines (IL-34, M-CSF, TGF-&#946;1, CD200, CX3CL1) for 6 days to facilitate microglial maturation.</li>
	<li>On day 23, collect the resulting neural organoids for characterization or further experiments.</li>
</ol>
4. Clearance and immunostaining of 3D neural organoids
<ol>
	<li>Fix up to 10 organoids by immersion in 1 mL of 4% paraformaldehyde (PFA) at 4 &#176;C for 24 h.</li>
	<li>Transfer the organoids per well to a 96-well clear bottom plate, and wash with 200 &#181;L of DPBS 2 times for 1 h each time with gentle shaking.</li>
	<li>Permeabilize organoids by washing them once in 50% methanol in DPBS, 80% methanol in double distilled (dd) water, and 100% dry methanol for 10 min each, at 4 &#176;C with gentle shaking.</li>
	<li>Wash the organoids serially in 20% DMSO/methanol, 80% methanol in dd water, 50% methanol in DPBS, in 100% DPBS, then in DPBS with 0.2% Triton X-100, for 10 min each at 4 &#176;C with gentle shaking. Make an effort to remove the residual buffer completely after each washing.</li>
	<li>Incubate in permeabilizing buffer (DPBS with 0.2% Triton X100, 0.3 M glycine, and 20% DMSO) with gentle shaking for 40 min at RT.</li>
	<li>Block in blocking buffer (DPBS with 0.2% Triton X100, 6% Goat serum, and 10% DMSO) for 1 h at 37 &#176;C with gentle shaking on a shaker at 80 rpm.</li>
	<li>Incubate with antibodies in antibody dilution buffer (IBA1 1:100, TREM2 1:100, &#946;III-tubulin, 1:1000, 100 &#181;L /well in DPBS with 0.2% Tween 20, 100 &#181;g/mL Heparin, 3% Goat serum and 5% DMSO) at 37 &#176;C for 2 h or in cold room with gentle shaking at 80 rpm for 3 days.</li>
	<li>Wash with DPBS with 0.2% Tween 20, 100 &#181;g/mL Heparin 5 times, 10 min each at RT with gentle shaking on a shaker at 80 rpm.</li>
	<li>Incubate with secondary antibody (1:200 goat anti-mouse Alexa488) at 37 &#176;C for 1 h followed by RT overnight with gentle shaking on a shaker at 80 rpm.</li>
	<li>For nuclear contrast staining, perform DAPI staining together with antibody incubation or in a washing buffer.</li>
	<li>Wash the organoids 10 times in washing buffer, 10 min each time at 37 &#176;C, with gentle shaking.</li>
	<li>Observe under a microscope. If the background is still high, keep washing it 5 times.</li>
	<li>Discard the supernatant as much as possible. Add 200 &#181;L of organoid clearing solution and incubate it for 5 min before observation and take images using a confocal microscope.</li>
</ol>

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

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</ol>

The authors have nothing to disclose.

Here, a detailed protocol for making 3D neural organoids containing innate microglia from EBs derived from mixed iPSCs and iPSC-differentiated HPCs is presented. It is a relatively short and easy approach involving only cell culture techniques and equipment generally available in most laboratories.
The most critical factor for the success of this protocol is the quality of HPC differentiation. We adopted the published method17 using a commercial kit to differentiate HPC...

Microglia are residential immune cells in the brain, playing critical roles in both brain development and homeostasis1,2. The activation of microglia results in the production of proinflammatory factors, elevated phagocytosis, and reactive oxidative stress, which removes the invading pathogens and compromised cells. However, overactivation or prolonged activation of microglia may, on the other hand, cause neurodegeneration as a mechanism of pathogenesis in many neurological disorders, including Parkinson&#39;s disease3,4. It is important that microglia are included in the relevant models for studying human neurological disorders. In recent years, human stem cells have been used to develop 3D organoids as in vitro models as an alternative to animal models and human subject studies5. Ideally, human organoids constitute multiple cell types and tissue structures similar to the corresponding human organs, better representing human physiology and pathogenesis than animal models but without the ethical concerns involved in studies of human individuals directly. They may represent the future of human disease modeling for the study of pathogenesis and drug development and for the guidance of individualized therapies6. As an example, 3D human brain organoids derived from human induced pluripotent stem cells (iPSCs) have prevailed in the field of neuroscience research, modeling neural infectious diseases including ZIKA, SARS-CoV-27, and neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer&#39;s disease8,9. However, conventional 3D neural organoids using dual-SMAD inhibition to induce neuronal differentiation10 produce brain organoids lacking microglia, as they are&#160;derived from progenitors recruited from the blood instead of the neuroectoderm lineage that neurons are from11,12. Without the presence of microglia, the organoids are inadequate to model CNS infections, inflammation, and the associated neurodegeneration.
To address this critical issue, attempts have been made to incorporate differentiated microglia into the brain organoids13 or inducing microglial differentiation within organoids from the beginning using alternative approaches instead of dual-SMAD inhibition13. However, by incorporating differentiated microglia into the brain organoids, the early interactions between neuronal and microglial development are missed. This could be important in CNS development or the pathogenesis of neuroinfectious disorders targeting infant brain development, such as in ZIKA virus infection14. On the other hand, differentiating innate microglia within iPSC-derived brain organoids without intermittent stages involves a prolonged process and has higher variability within the end products15. In this reported protocol, we incorporated the iPSC-derived hematopoietic&#160;progenitor cells (HPCs) into the iPSCs to make embryoid bodies (EBs), which were further differentiated into 3D organoids including both neurons and microglia.
Our protocol provides an easy approach that can be adopted to study the human central nervous system involving early neuron-microglial interactions and the pathogenesis of neural infectious disorders and neuroinflammation involving microglial activation.

<table><tbody><tr><td>12 well cell culture plates</td><td>Corning&amp;nbsp;</td><td>#3512</td><td></td></tr><tr><td>24 well cell culture plate</td><td>SARSTEDT</td><td>#83.3922</td><td></td></tr><tr><td>Accutase</td><td>Thermo</td><td>A1110501</td><td></td></tr><tr><td>Aggrewell 400 plate</td><td>Stemcell technologies</td><td>#34411</td><td>Referred to as microwell culture plate&amp;nbsp;</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse antibody</td><td>Life techniologies</td><td>A11001</td><td>1:400 dilution</td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit antibody</td><td>Life techniologies</td><td>A11012</td><td>1:400 dilution</td></tr><tr><td>Allegra X-30R Centrifuge with rotor S6069</td><td>Beckman Couler</td><td></td><td></td></tr><tr><td>Anti- Adherence&amp;nbsp; Rinsing solution</td><td>Stem Cell Technologies</td><td>#07010</td><td></td></tr><tr><td>anti-CD34 antibody</td><td>Stem Cell Technologies</td><td>#60013</td><td>1:100 dilution</td></tr><tr><td>anti-Human CD43 antibody</td><td>Stem Cell Technologies</td><td>#60085</td><td>1:100 dilution</td></tr><tr><td>anti-IBA1 rabbbit antibody</td><td>Fujifilm</td><td>019-19741</td><td>2.5 &amp;micro;g/mL</td></tr><tr><td>anti-TREM2&amp;nbsp; rat pAb</td><td>RD Systems</td><td>mab17291</td><td>2.5 &amp;micro;g/mL</td></tr><tr><td>Antibiotic-antimycotic</td><td>Gibco</td><td>15240-062</td><td>1x</td></tr><tr><td>B27 supplement</td><td>Life technologies</td><td>17504-044</td><td>1x</td></tr><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>20 ng/mL</td></tr><tr><td>CD200</td><td>Novoprotein</td><td>C311</td><td>&amp;nbsp;100 ng/mL</td></tr><tr><td>CryoTube vials</td><td>Thermo</td><td>#368632</td><td></td></tr><tr><td>CX3CL1</td><td>Peprotech</td><td>300-31</td><td>&amp;nbsp;100 ng/mL</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>1 &amp;micro;g/mL</td></tr><tr><td>DMEM/F12</td><td>Life technologies</td><td>12400-024</td><td>1x</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>#4190136</td><td>1x</td></tr><tr><td>E8 Flex medium kit</td><td>Thermo</td><td>A2858501</td><td></td></tr><tr><td>EDTA</td><td>Mediatech</td><td>46-034-Cl&amp;nbsp;</td><td>0.5 mM</td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-15</td><td>20 ng/mL</td></tr><tr><td>EVOS FL Auto Microscope</td><td>Thermo</td><td></td><td>Fluorescence microscope&amp;nbsp;</td></tr><tr><td>FastStart Universal SYBR Green PCR master mix</td><td>Roche</td><td>#4913850001</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>#35050079</td><td></td></tr><tr><td>Goat serum</td><td>Sigma</td><td>G9023</td><td>4%</td></tr><tr><td>IL-34</td><td>Peprotech</td><td>200-34</td><td>&amp;nbsp;100 ng/mL</td></tr><tr><td>ImageXpress Micro Confocal</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Knockout DMEM/F12</td><td>Gibco</td><td>#10829018</td><td></td></tr><tr><td>M-CSF</td><td>Peprotech</td><td>300-25</td><td>&amp;nbsp;25 ng/mL</td></tr><tr><td>Matrigel</td><td>Corning</td><td>#354277</td><td>Basement membrane matrix (BMM)</td></tr><tr><td>Mouse anti-&amp;beta;III-tubulin antibody</td><td>Promega</td><td>G712A</td><td>1:1000 dilution</td></tr><tr><td>Mr. Frosty container</td><td>Thermo</td><td>5100-0001</td><td></td></tr><tr><td>N2 supplement</td><td>Life technologies</td><td>17502-048</td><td>1x</td></tr><tr><td>Paraformadehyde</td><td>Sigma</td><td>P6148</td><td>4%</td></tr><tr><td>PSC Neural Induction Medium</td><td>Gibco</td><td>A1647801</td><td></td></tr><tr><td>Rock inhibitor Y27632</td><td>Stemcell technologies</td><td>#72304</td><td>1 mM stock</td></tr><tr><td>RT LTS 1000 ul pipette tips</td><td>RAININ</td><td>#30389218</td><td>&amp;nbsp;for transferring organoids</td></tr><tr><td>STEMdiff Cerebral Organoid Kit</td><td>Stem Cell Technologies</td><td>#08570</td><td></td></tr><tr><td>STEMdiff Hematopoietic Kit</td><td>StemCell Technologies</td><td>#5310</td><td>Referred to as hematopoietic Kit</td></tr><tr><td>StemPro Neural Supplement</td><td>Gibco</td><td>A1050801</td><td>Referred to as neural supplement</td></tr><tr><td>TGF-&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>50 ng/mL</td></tr><tr><td>Total RNA Purification Plus Kit</td><td>Norgen</td><td>#48400</td><td></td></tr><tr><td>TritonX-100</td><td>Sigma</td><td>T9284</td><td>0.10%</td></tr><tr><td>Visikol Histo-Starter Kit</td><td>Visikol</td><td>HSK-1</td><td>Contains organoid clearing solution HISTO-M, washing buffer</td></tr><tr><td>Zeiss LSM 510-META Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

derivation of a human brain organoid with microglia development

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

Our protocol follows a scheme to differentiate HPCs from iPSCs and then mix the HPCs with iPSCs to make EBs, followed by neural induction, differentiation, and maturation (Figure 1). High quality of HPC differentiation is critical for the success of EB formation and later organoid differentiation. A serial dilution culture technique is used to produce the appropriate numbers and size of iPSC colonies to start the HPC differentiation (Figu...

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

derivation-of-a-human-brain-organoid-with-microglia-development

Research

JoVE Journal

Developmental Biology

The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

An In Vitro Method to Generate Human Brain Organoids

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

This study introduces and describes protocols to derive two specific human neural organoids as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Obtaining Human Microglia from Adult Human Brain Tissue

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

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.

Assembling Retinal Organoids with Microglia

Learn to generate human brain organoids containing resident microglia and neurons using iPSC-derived hematopoietic progenitor cells. This method enables study of microglia-neuron interactions during brain development.

Derivation of a Human Brain Organoid with Microglia Development

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