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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 days)
<ol>
	<li>Initiation of neuroectoderm (5 days) 
	NOTE: The following points should be considered before the start of differentiation. The reprogramming method (lentiviral-, sendai-virus-, episomal-, or microRNA-based etc.) to obtain human iPSCs should ideally be the same for all patient and control iPSC lines. Various reprogramming kits and instructions based on published protocols are available. The quality of the human iPSC lines is the key to accomplishing an optimal differentiation. Monitor colony and cell morphology with a microscope and validate pluripotency by testing the expression of markers such as Oct3/4, Nanog, or TRA-1-60.
	<ol>
		<li>Culture human iPSCs under feeder-free conditions in medium A on a dish coated with Engelbreth-Holm-Swarm (EHS) matrix. 
		NOTE: Grow human iPSCs feeder-free and serum-free to maintain a defined culture condition and to avoid an additional step for the removal of mouse embryonic feeder cells (MEFs) before the start of differentiation. The optimal passage number for human iPSCs to start differentiation ranges from passage 15 upon reprogramming to passage 70 in total. When passaging, detach hiPSC colonies as cells aggregate using an appropriate cell detachment solution with low mechanical stress and a 2-mL serological pipette to transfer aggregates to a new dish. Avoid dissociation to single cells, as this might induce differentiation and apoptosis in most iPSC lines. It was reported that long-term, single-cell passaging could increase genomic alterations in human iPSCs. Avoid cell detachment procedures, which require a centrifugation step to remove detachment solution, as this reduces the overall viability of iPSCs. Test all cultures for microbial contaminations, particularly mycoplasma, on a regular basis, because this might alter the quality of the iPSCs and their differentiation capacity.
		<ol>
			<li>Coat a 60 mm tissue culture dish with EHS matrix as per the manufacturer&#39;s instructions.</li>
			<li>Thaw an aliquot of 1 x 106 human iPSCs. Seed the iPSCs in a 60-mm tissue culture dish coated in EHS matrix and containing 5 mL of medium A (see the Materials Table). Change the medium daily and passage after 5 to 7 days when the cells reach ~80% confluency. 
			NOTE: Passage the thawed iPSCs at 80% confluency using standard methods, such as the enzyme-free detachment of colonies, at least once before starting the differentiation. In brief, remove the iPSC medium, wash the cells once with pre-warmed 37 &#176;C Dulbecco&#39;s Modified Eagle&#39;s Medium: Nutrient Mixture F-12 (DMEM/F12), and incubate the iPSCs following the manufacturer&#39;s instructions using reagent A (see the materials table). Don&#39;t exceed the recommended incubation time in order to avoid dissociation to single cells. Human iPSCs should be detached and floating as aggregates, not as single cells. They can then be transferred to new EHS matrix-coated dishes; for example, iPSC aggregates from one 60-mm dish can be distributed to 4 new 60-mm dishes. 
			b. Check for mycoplasma with a mycoplasma detection kit according to the manufacturer&#39;s instructions. Use only mycoplasma-free iPSCs, as mycoplasma can alter the differentiation capability of iPSCs.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Dissociate iPSCs (80% confluent) and prepare a single-cell suspension using reagent B
	<ol>
		<li>Wash the iPSCs once with pre-warmed (37 &#176;C) DMEM/F12.</li>
		<li>Add pre-warmed reagent B (e.g., 1 mL in a 60 mm dish) and incubate the iPSCs for 5 min at 37 &#176;C and 5% CO2.</li>
		<li>Flick 20 times with a fingertip on the side and bottom of the dish to detach the iPSCs. Check for the detachment of cells under the microscope.</li>
		<li>Pipette the cell suspension up and down in the dish 5 times with a 1-mL micropipette.</li>
		<li>Add 3 mL of medium A to dilute 1 mL of reagent B and collect the cell suspension in a 15-mL centrifuge tube.</li>
		<li>Gently spin down the iPSCs (500 x g) for 4 min at room temperature.</li>
		<li>Resuspend the cell pellet in 1 mL of medium B and count the cell number with a hemocytometer. 
		NOTE: Be aware of using only medium B for resuspension. Avoid using medium A, as it contains a too-high concentration of bFGF, which might inhibit the differentiation.</li>
	</ol>
	</li>
	<li>Dilute the cell suspension to 4.5 x 105 cells per mL in medium B supplemented with 10 &#181;M rho-associated protein kinase inhibitor (Y-27632).</li>
	<li>Add 100 &#181;L per well in a non-adherent, v-bottom, 96-well plate. 
	NOTE: Make sure the cells are equally distributed in the suspension by shaking the tube each time before taking out 100 &#181;L portions. It is important that each well should contain an equivalent cell number in order to obtain neurospheres homogenous in size and shape (round, defined surfaces).</li>
	<li>Gently spin down the plate with the cells at 500 x g and room temperature for 3 min and incubate at 37 &#176;C and 5% CO2.</li>
	<li>Change the medium daily by removing 50 &#181;L and adding 50 &#181;L of fresh medium B into each well for the next 5 days.</li>
</ol>
2. Embedding Neurospheres in EHS Matrix (4 days)
<ol>
	<li>Prepare neurosphere medium by mixing the following: 1:1 mixture of DMEM/F12 and medium C (v/v), 1:200 (v/v) supplement 1, 1:100 (v/v) supplement 2 without (w/o) Vitamin A, 1:100 L-glutamine, 0.05 mM non-essential amino acids (MEM), 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 1.6 g/L insulin, and 0.05 mM &#946;-mercaptoethanol.</li>
	<li>Collect the neurospheres with a 200 &#181;L micropipette using a ~2 mm tip previously cut with sterile scissors.</li>
	<li>Place the neurospheres approximately 5 mm away from each other on paraffin film (3 x 3 cm2) in an empty 100 mm dish and carefully remove as much of the remaining medium as possible.</li>
	<li>Add a drop (7 &#181;L) of EHS matrix onto each single neurosphere.</li>
	<li>Incubate the EHS matrix drops with the neurospheres for 15 min in an incubator.</li>
	<li>Wash the neurospheres carefully from the paraffin film by flushing them with neurosphere medium. To flush, use a 1 mL micropipette and a new 100 mm Petri dish containing 10 mL of neurosphere medium.</li>
	<li>Incubate the neurospheres for the next 4 days and add 2 mL of fresh neurosphere medium on day 2. 
	NOTE: Make sure that the shelves in the incubator are flat so that the EHS matrix-embedded neurospheres will not clump together on one side of the dish.</li>
</ol>
3. Organoids in a Rotary Suspension Culture (14 Days)
<ol>
	<li>Prepare brain organoid medium by mixing the following: 1:1 mixture of DMEM/F12 and medium C (v/v), 1:200 (v/v) supplement 1, 1:100 (v/v) supplement 2 w/o Vitamin A, 1:100 L-glutamine, 0.05 mM MEM, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 1.6 g/L insulin, 0.5 &#181;M dorsomorphin, 5 &#181;M SB431542, and 0.05 mM &#946;-mercaptoethanol.</li>
	<li>Add 100 mL of brain organoid medium to each spinner flask through its side arms and place them in an incubator for pre-warming for at least 20 min.</li>
	<li>Set up a stirring program at 25 rpm, according to the manufacturer&#39;s instructions. 
	NOTE: Before transferring the EHS matrix-embedded neurospheres into spinner flasks, ensure they are all separated. If two or more are connected through the EHS matrix, separate them by cutting the connecting matrix with a scalpel.</li>
	<li>Carefully transfer the EHS matrix-embedded neurospheres into spinner flasks containing 100 mL of organoid medium using a 2 mL serological pipette. Use the side arms of the spinner flask to transfer the neurospheres into the flask.</li>
	<li>Place the spinner flasks on a magnetic stirring platform in an incubator at 37 &#176;C and 5% CO2; this is day 0 of the organoid culture.</li>
	<li>Change the medium once per week (or more often when there is a color change) by removing half of the medium and adding the same amount of fresh medium. 
	NOTE: When taking the spinner flasks out of the incubator, wait 3-5 min to let the organoids sink down to the bottom of the flask. Remove the medium by placing the glass pipette tip (connected to a pump) on the surface of the liquid; aspirate the medium carefully through one side opening/arm of the flask. These manipulations must be done under the laminar hood.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco, US</td>
			<td>31331093</td>
			<td>Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma-Aldrich, US</td>
			<td>P5499</td>
			<td>Compound C</td>
		</tr>
		<tr>
			<td>Embedding medium</td>
			<td>AppliChem</td>
			<td>A9011, 0100</td>
			<td>Mowiol; embedding medium; multiple suppliers</td>
		</tr>
		<tr>
			<td>Engelbreth-Holm-Swarm (EHS) matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Matrigel hESC-qualified matrix; important: hESC qualified</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich, US</td>
			<td>I3536-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco, US</td>
			<td>25030081</td>
			<td>L-glutamine (200 mM)</td>
		</tr>
		<tr>
			<td>Medium A</td>
			<td>Stem cell technologies</td>
			<td>#05850</td>
			<td>mTeSR1 (hiPSC medium)</td>
		</tr>
		<tr>
			<td>Medium B</td>
			<td>Stem cell technologies</td>
			<td>#05835</td>
			<td>Neural induction medium (NIM); neural differentiation medium</td>
		</tr>
		<tr>
			<td>Medium C</td>
			<td>Gibco, US</td>
			<td>21103049</td>
			<td>Neural Basal Medium</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Gibco, US</td>
			<td>11140035</td>
			<td>MEM non-essential amino acids solution (100x)</td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza, Switzerland</td>
			<td>#LT07-218</td>
			<td>Mycoplasma detection kit; multiple suppliers</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10.000 U/ml)</td>
			<td>Gibco, US</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent A</td>
			<td>Stem cell technologies</td>
			<td># 05872</td>
			<td>&#160;ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer&#180;s protocol</td>
		</tr>
		<tr>
			<td>Reagent B</td>
			<td>Sigma-Aldrich, US</td>
			<td>A6964-100ML</td>
			<td>Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 &#34;enzymatic cell dissociation solution&#34;</td>
		</tr>
		<tr>
			<td>Supplement 1</td>
			<td>Gibco, US</td>
			<td>17502048</td>
			<td>N-2 supplement (100x)</td>
		</tr>
		<tr>
			<td>Supplement 2 w/o Vitamin A</td>
			<td>Gibco, US</td>
			<td>12587010</td>
			<td>B-27 supplement (50x), minus vitamin A</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Selleckchem.com</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner flask 250 ml</td>
			<td>IBS Integra Bioscience</td>
			<td>182026</td>
			<td></td>
		</tr>
		<tr>
			<td>waterproof sheet</td>
			<td>BEMIS company, inc.</td>
			<td>PM996</td>
			<td>Parafilm &#34;M&#34;</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleckchem.com</td>
			<td>S1049</td>
			<td>ROCK-inhibitor (Y-27632 2HCL)</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco, US</td>
			<td>31350010</td>
			<td>2-mercaptoethanol (50 mM)</td>
		</tr>
	</tbody>
</table>

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.

An In Vitro Method to Generate Human Brain Organoids

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

Begin with a culture of neurospheres, free-floating clusters of neuroepithelial progenitor cells.
Transfer these neurospheres onto an embedding sheet.
Remove media. Add a drop of extracellular membrane matrix containing proteins and growth factors to each neurosphere.
Incubate for the matrix to solidify, embedding the neurospheres within it.
Now, transfer the matrix-embedded neurospheres using neurosphere media to a culture plate containing neurosphere media.
Incubate. The extracellular membrane matrix and its constituents facilitate neuroepithelial progenitor cell differentiation into neuroepithelial cells which proliferate and form fluid-filled lumens.
Post-incubation, transfer neurospheres into a spinner flask containing pre-warmed brain organoid media. Incubate with stirring for enhanced oxygen and nutrient diffusion, promoting cellular growth.
Growth factors and small molecule inhibitors in media promote neural differentiation and develop into cells of specific brain regions, forming mature brain organoids.

an-in-vitro-method-to-generate-human-brain-organoids

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

74 Aufrufe. Quelle: Gabriel, E., et al., Generierung von iPSC-abgeleiteten Organoiden des menschlichen Gehirns zur Modellierung früher neurologischer Entwicklungsstörungen. J. Vis. Exp. (2017) Das Video demonstriert eine Methode zur Erzeugung menschlicher Gehirnorganoide aus induzierten pluripotenten Stammzellen. Es wird gezeigt, wie die Neurosphäre eine neuroektodermale Schicht bildet, die die Bildung anderer Keimblätter unterdrückt. Die Neurosphären sind in eine 3D-Gelmatrix eingebettet und bilden...

Serie: Eine In-vitro-Methode zur Erzeugung von Organoiden des menschlichen Gehirns - Konzept

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generierung eines humanen iPSC-basierten Blut-Hirn-Barriere-Chips

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Forschung

JoVE Journal

Entwicklungsbiologie

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;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 neurale Organoide zur Untersuchung von Hirntumor und neurodegenerativen Erkrankungen

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Biotechnik

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.

Generierung menschlicher Gehirnorganoide für die mitochondriale Krankheitsmodellierung

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

An In Vitro Method to Generate Human Brain Organoids

Transkript

An In Vitro Method to Generate Human Brain Organoids