We thank Miriam B&#252;nning for technical support. We acknowledge support from the Deutsche Forschungsgemeinschaft (DFG) (PR1527/5-1 to A.P.), Spark and Berlin Institute of Health (BIH) (BIH Validation Funds to A.P.), the United Mitochondrial Disease Foundation (UMDF) (Leigh Syndrome International Consortium Grant to A.P.), University Hospital Duesseldorf (Forschungskommission UKD to A.P.), and the German Federal Ministry of Education and Research (BMBF) (e:Bio young investigator grant AZ 031L0211 to A.P.).Work in the laboratory of C.R.R. was supported by the DFG (FOR 2795 &#34;Synapses under stress&#34;, Ro 2327/13-1).

NOTE: The use of human iPSCs may require an ethical approval. iPSCs used in this study were derived from healthy control individuals following local ethical approval (#2019-681). All cell culture procedures must be performed under a sterile cell culture hood, carefully disinfecting all reagents and consumables before transferring under the hood. Human iPSCs used for differentiation should have a passage number below 50 to avoid potential genomic aberrations that may occur upon extensive culture. The pluripotent state of ...

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This paper describes the reproducible generation of human iPSC-derived brain organoids and their use for mitochondrial disease modeling. The protocol described here is modified based on a previously published work20. One major advantage of the present protocol is that it does not require the manual embedding of each organoid into a scaffolding matrix. In fact, the matrix solution is simply dissolved into the cell culture medium. Moreover, there is no need to employ expensive bioreactors, as organo...

Mitochondrial diseases represent the largest class of inborn errors of metabolism1. They are caused by genetic mutations disrupting different mitochondrial processes, including oxidative phosphorylation (OXPHOS)2, respiratory chain assembly, mitochondrial dynamics, and mitochondrial DNA transcription or replication3. Tissues with energy requirements are particularly affected by mitochondrial dysfunction4. Accordingly, patients with mitochondrial diseases typically develop early-onset neurological manifestations.
There are currently no treatments available for children affected with mitochondrial diseases5. A major hindrance for drug development of mitochondrial diseases is the lack of effective models recapitulating the human disease course6. Several of the currently studied animal models do not exhibit the neurological defects present in the patients7. Hence, the mechanisms underlying the neuronal pathology of mitochondrial diseases are still not fully understood.
Recent studies generated iPSCs from patients affected by mitochondrial diseases and used these cells to obtain patient-specific neuronal cells. For example, genetic defects associated with the mitochondrial disease, Leigh syndrome, have been found to cause aberrations in cellular bioenergetics8,9, protein synthesis10, and calcium homeostasis9,11. These reports provided important mechanistic clues on the neuronal impairment occurring in mitochondrial diseases, paving the way for drug discovery for these incurable diseases12.
Two-dimensional (2D) cultures, however, do not enable the investigation of the architectural complexity and regional organization of 3D organs13. To this end, the use of 3D brain organoids derived from patient-specific iPSCs14 may allow researchers to gain additional important information and thereby help to dissect how mitochondrial diseases impact the development and function of the nervous system15. Studies employing iPSC-derived brain organoids to investigate mitochondrial diseases are beginning to uncover the neurodevelopmental components of mitochondrial diseases. 
Spinal cord organoids carrying mutations associated with the mitochondrial disease, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome (MELAS), showed defective neurogenesis and delayed motor neuron differentiation16. Cortical organoids derived from patients with the mitochondrial disease, Leigh syndrome, showed reduced size, defects in neural epithelial bud generation, and loss of cortical architecture17. Brain organoids from Leigh syndrome patients showed that the disease defects initiate at the level of neural progenitor cells, which cannot commit to mitochondrial metabolism, causing aberrant neuronal branching and morphogenesis18. Thus, neural progenitors may represent a cellular therapeutic target for mitochondrial diseases, and strategies promoting their mitochondrial function may support the functional development of the nervous system.
The use of brain organoids might help uncover the neurodevelopmental components of mitochondrial diseases. Mitochondrial diseases are mainly considered as early-onset neurodegeneration5. However, neurodevelopmental defects are also present in patients affected by mitochondrial diseases, including developmental delay and cognitive impairment19. Patient-specific brain organoids may help address these aspects and elucidate how mitochondrial diseases may impact human brain development. Mitochondrial dysfunction could also play a pathogenetic role in other more common neurological diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease4. Hence, elucidating the impact of mitochondrial defects in neurodevelopment using brain organoids might also be instrumental for the study of those diseases. This paper describes a detailed protocol for generating reproducible brain organoids that can be used for conducting disease modeling of mitochondrial diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Affinity Designer</td>
			<td>Serif (Europe) Ltd</td>
			<td></td>
			<td>Layout software; Vector graphics editor</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-guinea pig</td>
			<td>Sigma Aldrich</td>
			<td>SAB4600033-250UL</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31571</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma Aldrich</td>
			<td>1397-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III (TUJ-1)</td>
			<td>Sigma Aldrich</td>
			<td>T8578</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Argon Laser</td>
			<td>Melles Griot</td>
			<td></td>
			<td>Any other Laser, e.g., diode lasers emitting 488 is fine, too</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A92902</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 with Vitamin A</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>3% in PBS, store solution at -20 &#176;C</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-0811</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Star cell culture 6 well plate</td>
			<td>Greiner-Bio-One</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically Defined Lipid Concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope C1</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Modular confocal microscope system</td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement membrane matrix, Phenol Red-free, LDEV-free</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Matrix component</td>
		</tr>
		<tr>
			<td>CyQUANT Cell Proliferation Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher</td>
			<td>31330038</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2660-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat Cy3</td>
			<td>Merck Millipore</td>
			<td>AP180C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Cy3</td>
			<td>Merck Millipore</td>
			<td>AP192C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Cy3</td>
			<td>Merck Millipore</td>
			<td>AP182C</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Q1Mc camera</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse 90i upright widefield microscope</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse E 600FN upright microscope</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ts2 Inverted Microscope</td>
			<td>Nikon Microsope Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-C1 Silver Version 3.91</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Imaging software for confocal microscope</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma Aldrich</td>
			<td>370-86-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Glasgow MEM</td>
			<td>Gibco</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Brand</td>
			<td>747715</td>
			<td>Inverted</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium-Neon Laser</td>
			<td>Melles Griot</td>
			<td></td>
			<td>Every other Laser, e.g., diode lasers emitting 594 is fine, too</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck</td>
			<td>H3149-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACell 240i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026331</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1:2500</td>
		</tr>
		<tr>
			<td>Image J 1.53c</td>
			<td>Wayne Rasband National Institute of Health</td>
			<td></td>
			<td>Image processing Software</td>
		</tr>
		<tr>
			<td>Injekt Solo 10 mL/ Luer</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser (407 nm)</td>
			<td>Coherent</td>
			<td></td>
			<td>Any other Laser, e.g., diode lasers emitting 407 is fine, too</td>
		</tr>
		<tr>
			<td>Map2</td>
			<td>Synaptic Systems</td>
			<td>No. 188004</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Maxisafe 2030i</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stemcell Technology</td>
			<td>85850</td>
			<td>iPSC medium</td>
		</tr>
		<tr>
			<td>Multifuge X3R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>10325804</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td># LT07-218</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle for single usage (23G x 1&#8221; TW)</td>
			<td>Neoject</td>
			<td>10016</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Aadvanced Research 3.2</td>
			<td>Nikon</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>Oligomycin A</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker Heidolph Unimax 1010</td>
			<td>Heidolph</td>
			<td>543-12310-00</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP Pen</td>
			<td>Sigma</td>
			<td>Z377821-1EA</td>
			<td>To draw hydrophobic barrier on slides.</td>
		</tr>
		<tr>
			<td>Papain Dissociation System kit</td>
			<td>Worthington</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>818715</td>
			<td>4% in PBS, store solution at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Pasteur pipette 7mL</td>
			<td>VWR</td>
			<td>612-1681</td>
			<td>Graduated up to 3 mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apo VC 20x / 0.75 air DIC N2&#160; &#8734;/0.17 WD 1.0</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Dry Microscope Objective</td>
		</tr>
		<tr>
			<td>Plan Apo VC 60x / 1.40 oil DIC N2 &#8734;/0.17 WD 0.13</td>
			<td>Nikon Microscope Solutions</td>
			<td></td>
			<td>Oil Immersion Microscope Objective</td>
		</tr>
		<tr>
			<td>Polystyrene Petri dish (100 mm)</td>
			<td>Greiner Bio-One</td>
			<td>664161</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tube with cell-strainer cap (5 mL)</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Glass Antifade Moutant</td>
			<td>Invitrogen</td>
			<td>P36980</td>
			<td></td>
		</tr>
		<tr>
			<td>Qualitative filter paper</td>
			<td>VWR</td>
			<td>516-0813</td>
			<td></td>
		</tr>
		<tr>
			<td>Rock Inhibitior</td>
			<td>Merck</td>
			<td>SCM075</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>83-794</td>
			<td></td>
		</tr>
		<tr>
			<td>S100&#946;</td>
			<td>Abcam</td>
			<td>Ab11178</td>
			<td>1:600</td>
		</tr>
		<tr>
			<td>SB-431542</td>
			<td>Cayman Chemical Company</td>
			<td>13031</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Heinz Herenz Hamburq</td>
			<td>1110918</td>
			<td></td>
		</tr>
		<tr>
			<td>SMI312</td>
			<td>Biolegend</td>
			<td>837904</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Merck/Sigma</td>
			<td>31437-1kg-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>3957</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate</td>
			<td>Applichem</td>
			<td>131965</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-17320</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Gibco/StemPro</td>
			<td>A1110501</td>
			<td>Reagent A</td>
		</tr>
		<tr>
			<td>Super Glue Gel</td>
			<td>UHU</td>
			<td>63261</td>
			<td>adhesive gel</td>
		</tr>
		<tr>
			<td>SuperFrost Plus</td>
			<td>VWR</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe for single usage (1 mL)</td>
			<td>BD Plastipak</td>
			<td>300015</td>
			<td></td>
		</tr>
		<tr>
			<td>TB2 Thermoblock</td>
			<td>Biometra</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 24 Well</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 6 Well</td>
			<td>Sarstedt</td>
			<td>83.392</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFbeta3</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>ThermoFisher</td>
			<td>51032711</td>
			<td></td>
		</tr>
		<tr>
			<td>TOM20</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-11415</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Merck</td>
			<td>X100-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Production terminated, any other adjustable microtome is fine, too.</td>
		</tr>
		<tr>
			<td>Vibratome Wilkinson Classic Razor Blade</td>
			<td>Wilkinson Sword</td>
			<td>70517470</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Benchkote</td>
			<td>Merck/Sigma</td>
			<td>28418852</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt Antagonist I</td>
			<td>EMD Millipore Corp</td>
			<td>3378738</td>
			<td></td>
		</tr>
		<tr>
			<td>XF 96 extracellular flux analyser</td>
			<td>Seahorse Bioscience</td>
			<td>100737-101</td>
			<td></td>
		</tr>
		<tr>
			<td>XF Assay DMEM Medium</td>
			<td>Seahorse Bioscience</td>
			<td>103680-100</td>
			<td></td>
		</tr>
		<tr>
			<td>XF Calibrant Solution</td>
			<td>Seahorse Bioscience</td>
			<td>100840-000</td>
			<td></td>
		</tr>
		<tr>
			<td>XFe96 FluxPak (96-well microplate)</td>
			<td>Seahorse Bioscience</td>
			<td>102416-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The protocol described here facilitates the robust generation of round organoids (Figure 1A). The generated organoids&#160;contain mature neurons that can be visualized using protein markers specific for axons (SMI312) and dendrites (microtubule-associated protein 2 (MAP2)) (Figure 1B). Mature organoids contain not only neuronal cells (MAP2-positive) but also glial cells (e.g., positive for the astrocyte marker ...

Watch this Scientific Journal Video about Generation of Human Brain Organoids for Mitochondrial Disease Modeling at JoVE.com

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

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 ...

This protocol aims to use brain organoids to study mitochondrial diseases. It was developed for generating reproducible brain organoids in the assessment of their mitochondrial properties. This method does not require expensive bioreactors and time-consuming embedding procedures.Therefore, it is easy to implement and upscale. This protocol makes it possible to generate reproducible brain organoids and to test their mitochondrial properties. This can lead to identification of interventional targets for untreatable mitochondria diseases.To begin, culture human iPSCs under feeder-free conditions in iPSC medium on coated six-well plates and keep them in a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Passage the iPSCs at 80%confluency using enzyme-free detachment medium in ratios ranging from one by four to one by 12. To increase cell survival, add 10 micromolar rho-associated protein kinase or ROCK inhibitor after each splitting.Dissociate the 80%iPSCs at day zero. Prepare cortical differentiation medium one or CDM1 and pre-warm it at room temperature before adding it to the cells. Wash the wells containing the iPSCs with PBS to remove dead cells and debris.Add 500 microliters of pre-warmed reagent A to each well and incubate for five minutes at 37 degrees Celsius. Check under the microscope to ensure cell detachment. Add one milliliter of iPSC medium to dilute reagent A to neutralize its activity.Use a 1, 000 microliter pipette to dissociate the cells by piping up and down and transfer the cell suspension to a 15 milliliter centrifuge tube. Gently centrifuge the iPSCs at 125 times G for five minutes at room temperature, then carefully aspirate the supernatant to avoid disturbing the cell pellet. Resuspend the pellet with one milliliter of CDM1 to obtain a single cell suspension and count the cell number.Prepare the seeding medium with 9, 000 iPSCs per 100 microliters in CDM1 supplemented with 20 micromolar ROCK inhibitor, three micromolar WNT catenin inhibitor or IWR-1, and five micromolar SB-431542. Add 100 microliters of seeding medium per well to a 96-well V bottom plate. Keep the plate in a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide.To generate neurospheres, on day one, observe that round cell aggregates with defined smooth borders are forming. Note the dead cells around the aggregates. Continue to culture in the incubator at 37 degrees Celsius and 5%carbon dioxide.On day three, agitate the plate by tapping on the sides three times to detach dead cells, then add 100 microliters of CDM1 supplemented with 20 micromolar ROCK inhibitor, three micromolar IWR-1, and five micromolar SB-431542 to each well. Keep the plate to the incubator at 37 degrees Celsius and 5%carbon dioxide. On day six, carefully remove 80 microliters of the supernatant medium from each well.Avoid touching the bottom of the well. Add 100 microliters of CDM1 supplemented with three micromolar IWR-1 and five micromolar SB-431542 to each well and incubate the plate. Repeat the previous step after every three days until day 18.On day 18, to transfer neurospheres, prepare CDM2 and add 10 milliliters to a 100 millimeter ultra low attachment cell culture plate. Use a 200 microliter pipette with the tip cut off to transfer the round neurospheres from the 96-well plate to the 100 millimeter ultra low attachment cell culture plate. Remove five milliliters of medium from the plate containing the neurospheres and add five milliliters of fresh CDM2.Place the plate on an orbital shaker at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Every three days, carefully aspirate the supernatant medium and replace it with fresh CDM2. Leave a small amount of the medium to prevent neurospheres from drying out.On day 35, prepare CDM3. Aspirate the medium from the plate and add 10 milliliters of cold CDM3. After changing the medium, place the plate back on an orbital shaker at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide.Change the medium every three to five days depending on the rate of growth as indicated by the color of the medium. On day 70, prepare CDM4. Use CDM4 medium until the desired age of organoids is reached.During this period, keep the plate on an orbital shaker set at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Change the medium every three to five days depending on the growth rate. Prepare 4%PFA solution and place it under a safety hood.Collect brain organoids and gently transfer them with a blunt tipped three milliliter plastic Pasteur pipette to a six-well plate filled with PFA. Keep the organoids in the PFA solution for one hour at room temperature. Carefully remove the PFA with a three milliliter plastic Pasteur pipette and wash the fixed organoids three times using PBS.Store the fixed organoids at four degrees Celsius in PBS until further use. The organoids generated using this protocol contain mature neurons that can be visualized using protein markers specific for axons and dendrites. Mature organoids contain not only neuronal cells, but also glial cells.Using sliced brain organoids analysis, it is possible to monitor SMI 312 positive axons and MAP2 positive dendrites or the S100 beta glial cells. Further, confocal images help to investigate the detailed distribution and organization of SOX2 positive neural progenitors with respect to beta-3 tubulin positive neurons. Brain organoids were stained for mitochondria-specific markers, such as translocase of outer membrane or TOM20.Bioenergetic profiling of brain organoids was performed by measuring both mitochondrial metabolisms using the oxygen consumption rate, or OCR, and the glycolytic metabolism using the extracellular acidification rate, or ECAR. Oligomycin causes a drop in the OCR profile and therefore identifies the OCR needed for ATP production. Upon oligomycin treatment, there may also be a compensatory increase in ECAR suggesting that the cells can upregulate glycolysis to prevent the metabolic stress.When transferring the neurospheres from the 96-well plate to a culture plate or during the fixing procedure, it is crucial to handle the organoids gently to avoid damaging them. Following the generation of brain organoids, multi-electrode array or calcium imaging would be ideal methods to test the functionality of the organoids.

generation-of-human-brain-organoids-for-mitochondrial-disease-modeling

Research

JoVE Journal

Neuroscience

5.7K visualizações.  Heinrich Heine University. Este protocolo visa usar organoides cerebrais para estudar doenças mitocondriais. Foi desenvolvido para gerar organoides cerebrais reprodutíveis na avaliação de suas propriedades mitocondriais. Este método não requer bioreatores caros e procedimentos de incorporação demorados.Portanto, é fácil de implementar e upscale. Este protocolo permite gerar organoides cerebrais reprodutíveis e testar suas propriedades mitocondriais. Isso pode levar à identificação de alvos interven...