This work was supported by the Netherlands Organ-on-Chip Initiative, an NWO Gravitation project (024.003.001) funded by the Ministry of Education, Culture and Science of the government of the Netherlands. D.C.B. thankfully acknowledges the financial support of Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT) in the form of a doctoral scholarship.

Patient studies were approved by the Institutional Review Board. All participants signed written informed consent and repository consent to allow their data and biospecimen to be repurposed. iPSC lines were generated following IRB and institutional guidelines. Figure 1 illustrates a schematic overview of the workflow of this protocol.
1. iPSC reprogramming and maintenance
<ol>
	<li>Collect 8 mL of the subject&#39;s blood (aged or disease patient; ages 65 and older) into cell preparation tubes (CPT) with sodium citrate or into EDTA or heparinized tubes.</li>
	<li>Centrifuge at 1,800 x g for 30 min at room temperature (RT) to collect pellet containing only peripheral blood and no serum15.</li>
	<li>Use specialized reprogramming vectors according to the manufacturer&#39;s protocol (see Table of Materials) to obtain iPSCs16.</li>
	<li>Culture iPSCs in feeder-free, lactose dehydrogenase elevating virus (LDEV)-free reduced growth factor basement membrane matrix coated 6-well culture plates in Essential 8 (E8) media (see Table of Materials) at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>Maintain iPSCs in E8 media for 3-4 days in order to avoid overcrowding or spontaneous differentiation.</li>
	<li>Validate the pluripotency of the iPSC lines using immunofluorescent markers (step 2) and test for mycoplasma (step 3).</li>
</ol>
2. Pluripotency staining
<ol>
	<li>To conserve solutions and antibodies, seed the cells on coated (step 1.4) 24-well culture plates 3-4 days prior to analysis. Aspirate the medium with a pipette and fix the cells with 4% formaldehyde in phosphate-buffered saline (1x PBS) for 15-20 minutes at RT.</li>
	<li>Wash the cells 3x with 1x PBS and permeabilize with 0.1% Triton-X 100 in 1% bovine serum albumin (BSA) in PBS for at least 15 min, but no more than 1 h at RT.</li>
	<li>Wash 3x with 1x PBS and block with 5% BSA in PBS for 30 min at RT.</li>
	<li>Wash again 3x with 1x PBS and add the antibodies Sox2 and Oct3/4 (1:100 dilution; see Table of Materials), and the nucleic stain DAPI in 1% BSA (1:4,000 dilution) overnight at 4 &#176;C . Wrap in aluminum foil to protect from light.</li>
	<li>Wash 3x with 1x PBS and leave the cells in PBS. Image with a fluorescence microscope at a 10-20x magnification. Ensure that the pluripotency markers Sox2 and Oct3/4 are localized in the nuclei of the cells17,18.</li>
</ol>
3. Mycoplasma testing
NOTE: Refer to the detection kit&#39;s protocol (see Table of Materials) for detailed assay execution and analysis steps. The detection kit provides the reagent, the substrate, and the assay buffer for mycoplasma testing.
<ol>
	<li>Prior to passaging cells or refreshing medium, collect 2-3 mL of cell culture medium in a centrifuge tube and pellet any cells or debris at 200 x g for 5 min at RT. Store the supernatant at 4 &#176;C for &#8804;5 days. Incubate the cells with the medium for at least 24 h to ensure a detectable signal.</li>
	<li>Add 100 &#181;L of cell supernatant to a fresh tube or well of a white-walled 96-well plate (opaque bottom recommended, see Table of Materials). Reconstitute the reagent and substrate in the assay buffer and equilibrate for 15 min at RT.</li>
	<li>Add 100 &#181;L of the reagent to the sample and incubate for 5 min at RT. Measure the luminescence with a luminometer (measurement #1).</li>
	<li>Add 100 &#181;L of the substrate to the sample and incubate for 10 min at RT. Measure the luminescence (measurement #2).</li>
	<li>Determine the mycoplasma contamination by the ratio of measurement #2 to #1. Refer to the kit&#39;s manual for interpretation of the results.</li>
</ol>
4. Microfilament preparation
<ol>
	<li>Begin the preparation of the poly (lactic-co-glycolic acid) (PLGA, see Table of Materials) microfilaments by fraying the suture strand with the blunt end of a scalpel. Spay the frayed fiber lightly with 70% ethanol.</li>
	<li>Under the microscope and using a ruler, begin cutting the PLGA fiber into fragments of 500 &#181;m to 1 mm long strands. Cut about 25 mm of the fiber in total. Keep the filaments in a 15 mL tube with a 1 mL antibiotic-antimycotic solution.
	<ol>
		<li>In the hood, dilute the fiber solution with 10 mL of DMEM/F-12 (Table 1). Vortex well to mix the solution. 
		&#8203;NOTE: Work in a cell culture flow hood in a sterile environment.</li>
	</ol>
	</li>
	<li>Add 20 &#181;L of the fiber solution to three embryoid body (EB) forming wells of a 96-well plate. Under brightfield microscopy, count and average the fibers per well. Dilute or concentrate to average 5-10 PLGA microfilaments per well. Prepare each well in this manner.</li>
	<li>The wells are now ready to be seeded with cells. Store the plate at room temperature until needed or at 4 &#730;C for the following day.</li>
</ol>
5. Embryoid Body (EB) formation
NOTE: All media and solutions must be warmed to RT.
<ol>
	<li>Once the iPSCs have reached 70%-80% confluency (Figure 2A), they are ready to be passaged and used for EB formation. Check the cells with a microscope at 10x-20x magnification. Ensure that the colonies display minimal (&#60;10%) areas of spontaneous differentiation.</li>
	<li>Aspirate the medium with a pipette and wash the cells once with DPBS. Dissociate the colonies by adding 500 &#181;L of cell detachment solution (see Table of Materials) or 0.5 mM of ethylenediaminetetraacetic acid (EDTA) and incubate for 3-5 min at 37 &#176;C. 
	NOTE: Work in a cell culture flow hood in a sterile environment.</li>
	<li>Collect the released cells by adding 1 mL of fresh E8 media to each well and pipette gently until all cells are detached.</li>
	<li>Transfer 1.5 mL of cell suspension into a 15 mL tube, and add another 1 mL of fresh E8 media to reach a total volume of 2.5 mL.</li>
	<li>Centrifuge at 290 x g for 3 min at RT.</li>
	<li>Aspirate the supernatant with a pipette, resuspend the cell pellet in 1 mL of Essential 6 (E6, see Table of Materials) media supplemented with 50 &#181;M ROCK inhibitor, and count the cells using a hemocytometer19.</li>
	<li>Prepare a cell suspension of 60,000-90,000 cells/mL, depending on the desired seeding density, in E6 media supplemented with 50 &#181;M of ROCK inhibitor (see Table of Materials).</li>
	<li>Add 150 &#181;L of the cell suspension into each well of a 96-well ULA plate (or a prepared concave plate20). Seed 9,000-11,000 cells per well.</li>
	<li>Centrifuge the plate to force-aggregate the cells at 290 x g for 1 min at RT. Place the plate in the incubator at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
</ol>
6. Neuroepithelial induction
<ol>
	<li>After 24 h, carefully aspirate 120 &#181;L of the media with a pipette. Ensure not to aspirate the EB by lowering the pipette tip too far into the well.</li>
	<li>Add 150 &#181;L of E6 media (at RT) supplemented with 2 &#181;M of XAV939 and the SMAD inhibitors: 10 &#181;M of SB431542 and 500 nM of LDN 193189 per well (see Table of Materials).</li>
	<li>Change the medium daily with freshly prepared E6 media supplemented with 2 &#181;M of XAV939, 10 &#181;M of SB431542, and 500 nM of LDN 193189. 
	NOTE: By day 6 (DIV6), the EBs should have a diameter of 550-600 &#181;m and be ready for further differentiation.</li>
</ol>
7. Organoid differentiation and maturation
NOTE: All media needs to be warmed to RT.
<ol>
	<li>At approximately DIV7, check whether all the EBs have reached a diameter of 550-600 &#181;m and display a smooth and clear edge (Figure 2B); at this stage, they are ready to be embedded in an extracellular matrix (ECM). 
	NOTE: Work in a sterile environment.</li>
	<li>Prepare dimpled embedding sheets from the thermoplastic sealing film (see Table of Materials) by placing a film sheet (about 4 in long) on an empty P200 box. Using a 15 mL conical tube or a 500 &#181;L microcentrifuge tube, gently press down the film sheet into the holes to make 12 dimples. Spray the film sheet with 70% ethanol and let it dry inside the flow hood with the UV light switched on for at least 30 min.</li>
	<li>Thaw a sufficient quantity of basement membrane matrix (Matrigel, see Table of Materials) on ice and place it inside the flow hood. 
	NOTE: Per EB, approximately 30 &#181;L of undiluted membrane matrix is needed. Always keep the membrane matrix below 4 &#176;C to prevent it from gelling. Pre-chilled pipette tips are also recommended as they decelerate the matrix from polymerizing in the tip during pipetting, thereby reducing material loss.</li>
	<li>Using a wide-bore P200 tip, transfer one EB to each dimple, and remove as much media as possible with a normal pipette tip. Be mindful not to let the EBs dry. Using a regular P200 tip, add ~30 &#181;L of undiluted membrane matrix to each organoid, ensuring that the EB is at the center of the droplet.</li>
	<li>Once all EBs are embedded in the matrix, place the film sheet containing the EBs in a sterile Petri dish. 
	NOTE: Optionally, a small Petri dish filled with sterile water can be placed in the larger Petri dish next to the film sheet to prevent evaporation.
	<ol>
		<li>Transfer the dish to an incubator and incubate at 37 &#730;C in a humidified atmosphere with 5% CO2 for approximately 10 min to let the membrane matrix solidify.</li>
	</ol>
	</li>
	<li>For each set of 12 embedded organoids, prepare 5 mL of differentiation media with B27 without vitamin A (Table 1) in one well of a ULA 6-well plate. Pre-warm the plate to 37 &#176;C in an incubator.</li>
	<li>Once the incubation time is finished, transfer the embedded EBs to the ULA 6-well plate by taking the film sheet and pushing out the dimples from the back of the sheet. If necessary, take 1 mL from the well and pipette it onto the sheet to help the droplets detach from the film. 
	NOTE: Important morphological changes can be seen 1 day after embedding; EBs go from having smooth edges to bulging protrusions forming buds (Figure 2C).</li>
	<li>After 2 days (DIV9), perform a half-media change. Be careful not to aspirate or damage the membrane matrix droplets in the process.</li>
	<li>After 2 more days (DIV11), perform a full media change, supplementing the media with 3 &#181;M of CHIR99021 (see Table of Materials).</li>
	<li>At DIV14, change the media to differentiation media with B27 with vitamin A (Table 1) for a gradual increase in organoid size.</li>
	<li>At DIV16, place the well-plate on an orbital shaker at 90 rpm inside an incubator. Change the media every 2 days.</li>
	<li>Every 40 DIV, dilute 500 &#181;L of the membrane matrix for every 50 mL of media for additional nutrients in the media.</li>
</ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+organoid+culture+without+Matrigel.">Towards organoid culture without Matrigel.</a> Communications Biology. 4 (1), 1-15 (2021).</li><li>Kaiser, A., Kale, A., Novozhilova, E., Olivius, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+Matrigel&#174;+on+the+survival+and+differentiation+of+a+human+neural+progenitor+dissociated+sphere+culture.">The effects of Matrigel&#174; on the survival and differentiation of a human neural progenitor dissociated sphere culture.</a> The Anatomical Record. 303 (3), 441-450 (2020).</li><li>Kakni, 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=Intestinal+organoid+culture+in+polymer+film-based+microwell+arrays.">Intestinal organoid culture in polymer film-based microwell arrays.</a> Advanced Biosystems. 4 (10), 2000126 (2020).</li><li>Lancaster, 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=Guided+self-organization+and+cortical+plate+formation+in+human+brain+organoids.">Guided self-organization and cortical plate formation in human brain organoids.</a> Nature Biotechnology. 35 (7), 659-666 (2017).</li><li>Tejchman, A., Zn&#243;j, A., Chlebanowska, P., Fr&#261;czek-Szczypta, A., Majka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+fibers+as+a+new+type+of+scaffold+for+midbrain+organoid+development.">Carbon fibers as a new type of scaffold for midbrain organoid development.</a> International Journal of Molecular Sciences. 21 (17), 5959 (2020).</li></ol>

The authors have nothing to disclose.

The standardized formation of EBs is a critical step in the reproducible conversion of pluripotent stem cells into brain organoids. The force aggregation of stem cells into singular tissues can vary depending on the geometry of the concave wells, seeding density, and well treatment. Although the current method cites a diameter range of 500-600 &#181;m after 6 days, these diameters do not exclude other diameters of proper organoid formation, as many other diameters have proven successful. However, varying diameters have been shown to influence differentiation rates and success29. For reproducibility purposes, diameter variations below 50 &#181;m are highly recommended20. Additionally, the number of neural induction days can be extended from 6 to 10 days to allow for the EBs to grow in diameter, as the use of SMAD inhibitors has been shown to efficiently produce and maintain neuroepithelia formation after only 5 days of exposure30. In the absence of SMAD inhibitors, neural induction can yield inconsistent results, requiring longer induction periods. SMAD inhibitors cited in this work are the most effective, but other dorsomorphin and TGF-&#946; small molecules can be used at their effective concentration.
The basement membrane matrix provides an excellent substrate for growth and can be administered differently. Early uses of the matrix included the basal membrane as a well coating31. However, the use of the matrix for embedding tissue showed to improve differentiation and maturation in these tissues32. For organoids, the use of the matrix has been shown to improve EB differentiation into organoids, promote maturation, and extend culture periods33. While organoids can still be formed without the matrix, as many groups have strived to accomplish a matrix-free tissue culture34,35, studies have found that the matrix-embedded organoids have a higher chance of prolonged culture times and require less maintenance36. The dimple method of embedding provides equal coverage of the organoid surface, ensuring similar diffusion, access to nutrients, and reproducible differentiation. Alternatively, dome embedding can be employed to fully encapsulate the organoids37.
The transfer of the EBs into the membrane matrix dimples is a critical step. Although a 1 mL pipette tip has a wide enough opening to transfer the EBs, a 200 &#181;L tip is preferred for ease of transfer. The 200 &#181;L tips need to be cut to create a large enough opening, ensuring a smooth edge to reduce shear stress when pipetting. Alternatively, wide-bore 200 &#181;L tips exist for ease of transfer. Pipetting needs to be done slowly, as fast pipetting might disrupt the organoid periphery and hinder proper growth. Special attention must be taken to ensure sufficient medium is transferred to provide nutrients. Too little medium runs the risk of drying out the organoid, causing necrosis. Transferring the EB with too much culture media can cause the matrix to be too diluted and fail to encapsulate the organoid securely. Ideally, the matrix must not be diluted by more than 50% to ensure its polymerization and function as an ECM for the EBs. If the embedding is unsuccessful, the EB can be recovered and re-encapsulated. Re-encapsulation in the fresh matrix can be done at any point to provide the organoid with additional support.
Similar to matrix embedding for scaffolding, PLGA fibers provide additional support for three-dimensional growth. Originally incorporated to produce elongated organoids and increase surface area38, the incorporation of PLGA fibers has progressively been identified as an additional tool to improve organoid differentiation and maturation39. As more labs look to reduce the use of the matrix or abolish it altogether, the self-organizing properties of organoids supported by the incorporated fibers provide enough scaffolding for three-dimensional tissue creation and differentiation39. Here, both methods were combined to increase the chances of long-term culture38,39. The incorporation of fibers during the initial aggregation is critical, as these may not be introduced at a later stage. After centrifugation, checking that a couple of fibers are among the aggregated cells will ensure that a fiber is incorporated in the EB. If not successful, a gentle aspiration of the well and re-centrifugation should ensure mixing.
Another critical step in organoid maintenance is the introduction of an orbital shaker for better media perfusion. In early iterations of organoid protocols, a spinning bioreactor was used to create agitation11. An orbital shaker at 90 rpm provides sufficient agitation without destroying the matrix droplet or damaging organoid morphology. Some groups refrain from using the matrix scaffold but retain shaker agitation to provide a suitable environment34. As with all protocols, the speed of the rotations must be adjusted depending on the shaker to reduce the amount of shear stress. If a dome embedding was chosen, a tilted shaker could be employed to reduce the amount of shear stress11,34.
Taken together, the integration of several selected techniques provides a robust method of iPSC-derived organoid formation. There are several ways of creating and maintaining organoids, but many of them focus on early differentiation trajectories. In this work, multiple different techniques were combined to culture organoids for extended periods of time, past the differentiation phase, and into a maturation period where aging phenotypes can start to develop. Incorporating these techniques allows for prolonged maturation without the need for exogenous biological factors to maintain the cultures, retaining the self-organization and natural progression of aging.

Aging disease models have become increasingly relevant as human life expectancy continues to rise. Large-scale genomic studies have uncovered aged populations with dysregulation of molecular processes and genetic changes affecting the quality of life1. The aging process is characterized by a general loss of functionality of the organism, including loss of cognitive function, increased risk for neurodegenerative disorders, and a host of chronic diseases2.
Current cell culture techniques do not appropriately represent the multifactorial nature of aging, as these dysfunctions cannot be properly replicated by using mutations, toxins, or infections3. Animal models that explore the process of aging are often associated with long experimental times and high costs, but also bring ethical considerations with them. Using induced pluripotent stem cells (iPSCs) from patients can elucidate the molecular mechanisms that underlie disease progression as iPSCs allow for the natural development of cells into mature tissue3. iPSCs have become the working horse of many labs investigating neurodegenerative disorders, as the cellular reprogramming of harvested cells does not seem to erase the disease or aging imprints of the donors4. These imprints recapitulate cellular phenotypes that have been demonstrated in human and animal models, making iPSCs suitable for examining individual cellular deterioration of the highly dense brain tissue5,6. IPSC-derived organoids have become the preeminent model for three-dimensional culturing of tissue, enabling more complex cell-to-cell interactions and an improved developmental recapitulation. Although primarily used for developmental data, organoids have been increasingly applied toward disease modeling, specifically models for inflammation, neurodegeneration, and aging7. Building upon previous iPSC studies, organoids retain disease phenotypes and cellular phenotypes within the physiological context of tissue-like network connections8,9. However, culturing three-dimensional tissue of certain dimensions can be challenging, especially for extended periods of time.
This work presents a detailed method for the reproducible generation of cerebral organoids that allows the tissue to mature substantially in size for longer periods. Cerebral organoid creation has remained relatively standardized, adopting methods from several prominent protocols10,11. However, several modifications have been suggested for improved differentiation and maintenance. These alternate methods include using neurogenic factors to enhance neural differentiation12, additional scaffolds for improved nutrient exchange promoting cellular longevity13, and low-sheer stress agitation for prolonged culture and growth14. These improvements have been incorporated into this method to develop mature organoids capable of expressing neurodegenerative and aging phenotypes.

<table><tbody><tr><td>2-&amp;beta;-Mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>31350010</td><td></td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindoledihydrochloride (DAPI)</td><td>Invitrogen</td><td>D1306</td><td>1:4000</td></tr><tr><td>6-well Clear Flat Bottom CELLSTAR Cell Culture Multiwell Plate&amp;nbsp;</td><td>Greiner Bio-One</td><td>657185</td><td></td></tr><tr><td>6-well Clear Flat Bottom Ultra-Low Attachment Well Plate</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>96-well Clear Round Bottom Ultra-Low Attachment Microplate</td><td>Corning</td><td>7007</td><td></td></tr><tr><td>96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td><td>Thermo Fisher Scientific</td><td>136101</td><td></td></tr><tr><td>Accutase</td><td>Sigma-Aldrich</td><td>46964</td><td>cell detachment solution&amp;nbsp;</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>B27 Suppement (with Vitamin A) (50x)</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>B27 Supplement (minus Vitamin A) (50x)</td><td>Gibco</td><td>12587010</td><td></td></tr><tr><td>BD&amp;nbsp;Vacutainer&amp;trade; Glass Mononuclear Cell Preparation (CPT) Tubes</td><td>Thermo Fisher Scientific</td><td>02-685-125</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td>With plate holders</td></tr><tr><td>CHIR99021</td><td>Selleck Chemicals</td><td>S2924</td><td></td></tr><tr><td>CytoTune Sendai Reprogramming Vector</td><td>Thermo Fisher Scientific</td><td>A1378001</td><td></td></tr><tr><td>ddPCR primers | human | MAPT</td><td>Bio-Rad</td><td>dHsaCPE192234</td><td></td></tr><tr><td>ddPCR primers | human | RBFOX3 (NeuN)&amp;nbsp;</td><td>Bio-Rad</td><td>dHsaCPE5052108</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo Fisher Scientific</td><td>11320074</td><td></td></tr><tr><td>Doublecortin (DCX)</td><td>Santa Cruz Biotechnology</td><td>SC-8066</td><td>1:500</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190144</td><td>no calcium, no magnesium</td></tr><tr><td>Eppendorf cups, 1.5 mL&amp;nbsp;</td><td>Eppendorf</td><td>0030 125.215</td><td></td></tr><tr><td>Essential 6</td><td>Gibco</td><td>A1516401</td><td></td></tr><tr><td>Essential 8</td><td>Gibco</td><td>A1517001</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)&amp;nbsp;</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>Falcon tubes, 15 mL, conical</td><td>Greiner Bio-One</td><td>188271-N</td><td></td></tr><tr><td>Formaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>252549</td><td>37% Stock solution, diluted to 4% in PBS</td></tr><tr><td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td><td>Thermo Fisher Scientific</td><td>A1413202</td><td></td></tr><tr><td>GlutaMax (100x)</td><td>Gibco</td><td>35050038</td><td></td></tr><tr><td>Hemacytometer cell counter</td><td>Hausser scientific</td><td>1490</td><td></td></tr><tr><td>HEPES Buffer</td><td>Thermo Fisher Scientific</td><td>15-630-080</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I9278</td><td></td></tr><tr><td>LDN 193189</td><td>StemCell Technologies</td><td>72147</td><td></td></tr><tr><td>MAP2</td><td>Abcam</td><td>ab32454</td><td>1:200</td></tr><tr><td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free</td><td>Corning</td><td>356230</td><td>basement membrane matrix</td></tr><tr><td>MEM-Non Essential Amino Acid Solution (MEM-NEAA; 100x)</td><td>Thermo Fisher Scientific</td><td>11140050</td><td></td></tr><tr><td>Multilabel Counter Victor 3 Plate Reader</td><td>Perkin Elmer&amp;nbsp;</td><td>1420</td><td>luminometer</td></tr><tr><td>MycoAlert Mycoplasma Detection Kit</td><td>Lonza</td><td>LT07-318</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td></td></tr><tr><td>NeuN</td><td>Millipore</td><td>MAB377</td><td>1:500</td></tr><tr><td>Neurobasal Medium</td><td>Thermo Fisher Scientific</td><td>21103049</td><td></td></tr><tr><td>Oct-3/4 Antibody (C-10) Alexa Fluor 647</td><td>Santa Cruz Biotechnology</td><td>sc-5279 AF647</td><td>1:100</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td>thermoplastic film sheet</td></tr><tr><td>PAX6</td><td>Thermo Fisher Scientific</td><td>42-6600</td><td>1:200</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Poly(lactic-co-glycolic acid) (PLGA) microfilaments</td><td>Ethicon</td><td>J463</td><td></td></tr><tr><td>QX200 Droplet digital PCR system</td><td>Bio-Rad</td><td>1864001</td><td></td></tr><tr><td>ROCK inhibitor (Y27632)</td><td>Selleck Chemicals</td><td>S1049</td><td></td></tr><tr><td>SB431542&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>1614/50</td><td></td></tr><tr><td>SOX2 Monoclonal Antibody (Btjce), Alexa Fluor 488, eBioscience</td><td>Invitrogen</td><td>53-9811-80</td><td>1:100</td></tr><tr><td>Synapsin I (SYN)</td><td>Calbiochem</td><td>574777</td><td>1:200</td></tr><tr><td>Triton-X 100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>TUJ1</td><td>Santa Cruz Biotechnology</td><td>sc-80005</td><td>Beta-3-tubulin; 1:500</td></tr><tr><td>XAV939</td><td>Tocris Bioscience</td><td>3748</td><td></td></tr></tbody></table>

robust tissue fabrication for long-term culture of ipsc-derived brain organoids for aging research

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent development of procedures describing the generation of human cerebral organoids, derived from human induced pluripotent stem cells (iPSCs), has the potential to fundamentally transform the ability to model and understand the aging of the human brain and related pathogenic processes. Here, an optimized protocol for generating, maintaining, aging, and characterizing human iPSC-derived cerebral organoids is presented. This protocol can be implemented to generate brain organoids in a reproducible manner and serves as a step-by-step guide, incorporating the latest techniques that result in improved organoid maturation and aging in culture. Specific issues related to organoid maturation, necrosis, variability, and batch effects are being addressed. Taken together, these technological advances will allow the modeling of brain aging in organoids derived from a variety of young and aged human donors, as well as individuals afflicted with age-related brain disorders, allowing the identification of physiologic and pathogenic mechanisms of human brain aging.

Integrating PLGA fibers, dimple embedding, and agitation leads to a robust generation of cerebral organoids that enables iPSC-derived cultures to be maintained for extended periods of time (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64586/64586fig01.jpg" /> 
Figure 1: Schematic illustration of the workflow and timeline of this method. <a href="https://www.jove.com/files/ftp_upload/64586/64586fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The initial neural induction period is comparable to previously published procedures11. The embryoid bodies (EB) begin as circular aggregates (Figure 2B) with white or transparent edges. As the EB is changed from neural induction media to neural maintenance media at DIV7, protrusions and buddings emerge within 24 h from the circular tissue (Figure 2C). Furthermore, as the organoid continues to grow, the surface area of the PLGA fiber helps the organoid to elongate (Figure 2D). During maturation, the edges of the organoid need to remain intact, as it is a good sign of healthy cells and development; otherwise, additional nutrients must be provided13 (Figure 2E). Growth of the organoids is further facilitated by the agitation of the organoid culture, as this improves perfusion with nutrients.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64586/64586fig02.jpg" /> 
Figure 2: Differentiation and maturation of cerebral organoids. (A) Representative image of an iPSC culture at 70%-80% confluency. (B) Embryoid bodies (EBs) were generated and neuroepithelial formation was induced until DIV7. EBs were then embedded in the membrane matrix and further differentiated toward cerebral organoids. (C) Organoids at DIV10 displaying distinct budding formations. Organoids can be further matured in the membrane matrix using either (D) dimple or (E) sandwich embedding, here shown at DIV30. (F) Long-term culture shows cerebral organoids growing to significant sizes (DIV70). Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64586/64586fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The dimensions and external morphology of the organoid are complemented by a complex architecture inside of the organoid. After fixing the tissue at DIV7 after the first stage of differentiation, cells of the EB express SOX2, an HMG box transcription factor that acts as a marker for multipotent neural stem cells21,&#160;as well as paired protein box Pax-6, indicating neural progenitor cells (Figure 3). Immature neurons marked by TuJ1 (class III &#946;-tubulin)22 can already be seen scattered throughout the tissue.
At this stage, an example of the self-organization that these organoids go through becomes apparent. The organization of radial structures, called rosettes, is analogous to the neural tube, with SOX2+ cells in the rosette center21 and PAX6 towards the rosette periphery. These rosettes give rise to neurons as they migrate out. These radiating cells are initially double positive for the neural stem/progenitor cell marker Nestin23 and glial fibrillary acidic protein (GFAP), similar to the radial glia found in neurogenic areas of the in vivo brain. As these migrating neurons mature, the cytoskeletal markers reflect this change22. The early-stage neural differentiation marker TuJ122 is visible in the inner circle of the rosette and shifts to microtubule-associated protein 2 (MAP2)24, a neuron-specific maturation marker at the periphery.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64586/64586fig03.jpg" /> 
Figure 3: Embryoid bodies at DIV7 show structural organization and immature characterization. Whole mount immunohistochemistry image of an EB at DIV7. (A)&#160;A composite image of the EB displaying (B)&#160;SOX2+ neural rosettes, (C) immature neurons (TUJ1) scattered throughout the EB, (D)&#160;neural progenitor cells (PAX6), and (E)&#160;nuclei visualized by DAPI. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64586/64586fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As the organoids age, the organization and markers of the developing neurons start replicating physiological conditions. At DIV30, many rosettes giving rise to neurogenic regions equivalent to the developing brain can be observed25. By DIV60, these SOX2+ neurogenic regions are non-existent and are replaced by mature MAP2 and NeuN26, a neuron differentiation marker, and positive neurons (Figure 4 and Figure 5). 
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64586/64586fig04.jpg" /> 
Figure 4: Organoids at DIV120 show mature neuronal characterization. Immunohistochemistry image of an organoid section at DIV 120. (A) A composite image of the section showing mature neuronal markers of (B)&#160;MAP2 (purple) and (C) NeuN (green). (D) Nuclei were visualized by DAPI. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64586/64586fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64586/64586fig05.jpg" /> 
Figure 5: Digital droplet PCR of DIV120 organoids. Digital droplet PCR graphs showing the absolute expression value of (A) MAP2 (top, blue) and (B) NeuN (bottom, green). Slashed yellow lines separate different iPSC lines (A, B, C, etc.), and organoids have been batched together. N = 5. <a href="https://www.jove.com/files/ftp_upload/64586/64586fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
These cytoskeletal markers can be used in conjunction with other post-mitotic markers (like doublecortin27 and synapsin28) to probe for synaptic plasticity and other age-related declines (Figure 6), as well as additional brain tissue like astrocytes and glia (Figure 7).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64586/64586fig06.jpg" /> 
Figure 6: Example of synaptic terminal analysis. Immunohistochemistry image of a section of the organoid at DIV 120. (A) A composite image of the section stained for (B)&#160;MAP2, (C)&#160;doublecortin (DCX), and (D)&#160;synapsin I (SYN). (E)&#160;Nuclei were visualized by DAPI. Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64586/64586fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64586/64586fig07.jpg" /> 
Figure 7: Evidence of glial development. Immunohistochemistry image of a section of the organoid at DIV 120. (A) A composite image of the section stained for (B) ionized calcium-binding adaptor molecule 1 (Iba1) and (C) NeuN. (D)&#160;Nuclei were visualized by DAPI. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64586/64586fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final Concentration</td>
			<td>Volume (50 mL total)</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>50%</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>50%</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>1x</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>B27 Supplement -/+ Vitamin A (50x)</td>
			<td>0.5x</td>
			<td>0.5 mL&#160;</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>0.25%</td>
			<td>12.5 &#181;L</td>
		</tr>
		<tr>
			<td>GlutaMAX (100x)</td>
			<td>1x</td>
			<td>0.5 mL&#160;</td>
		</tr>
		<tr>
			<td>MEM-NEAA (100x)</td>
			<td>0.5x</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>10 mM</td>
			<td>0.5 mL&#160;</td>
		</tr>
		<tr>
			<td>Antibiotic/Antimycotic (100x)</td>
			<td>1x</td>
			<td>0.5 mL&#160;</td>
		</tr>
		<tr>
			<td>2-&#946;-mercaptoethanol</td>
			<td>50 &#181;M</td>
			<td>17.5 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">*NOTE: some DMEM-F12 already contains GlutaMAX, no need to add additional.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the differentiation media used in the present study.

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Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis.

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent ...

robust-tissue-fabrication-for-long-term-culture-ipsc-derived-brain

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Neuroscience

3.2K Views.  University of Twente. The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis. 

Video: Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

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

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

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.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Developmental Biology

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

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

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.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

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

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic 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.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

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

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

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.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

Cancer Research

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

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.

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 ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

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

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

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

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.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

An In Vitro Method to Generate Human Brain Organoids

Source: Gabriel, E., et al., <a href="https://app.jove.com/v/55372">Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.</a> J. Vis. Exp. (2017)
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.

Metoda in vitro do generowania organoidów ludzkiego mózgu

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 ...

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

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

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

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

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

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Derivation of a Human Brain Organoid with Microglia Development

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

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

Metoda in vitro do generowania organoidów ludzkiego mózgu