eoe sub articles

EoE Sub Articles

1. Seeding of human iPSCs in the bioreactor
<ol>
	<li>Incubate iPSCs grown as monolayers in mTeSR1 supplemented with 10 &#181;M of ROCK inhibitor Y-27632 (ROCKi). Add 1 mL of supplemented medium to each well from a 6-well tissue culture plate and incubate for 1 h at 37 &#176;C, 95% humidity, and 5% CO2. 
	NOTE: ROCKi is used to protect dissociated iPSCs from apoptosis.</li>
	<li>After 1 h of incubation, aspirate the spent medium from each well and wash 1x with 1 mL of 1...

generating mature cerebellar organoids from induced pluripotent stem cells using single-use bioreactors

Source: Silva, T.P., et al. <a href="https://app.jove.com/v/61143">Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for generating mature cerebellar organoids from induced pluripotent stem cells (iPSCs) using a single-use 3D bioreactor. The bioreactor facilitates iPSC aggregate formation, which then undergoes treatment with a series of culture media enriched in growth factors and chemokines that induce the differentiation of the iPSC aggregates into mature cerebellar organoids. Subsequently, the organoids are harvested and cryopreserved for further analysis.

Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors

1. Seeding of human iPSCs in the bioreactor

	Incubate iPSCs grown as monolayers in mTeSR1 supplemented with 10 &#181;M of ROCK inhibitor Y-27632 (ROCKi). ...

Seed human induced pluripotent stem cells, or iPSCs, in a bioreactor. The iPSCs are resuspended in media containing a cellular apoptosis inhibitor.
Under a magnetic field, the vertical wheel of the bioreactor rotates at a controlled speed, facilitating cell-cell contact and aggregate formation. Remove most of the media and add fresh media without the inhibitor.
Continue culturing. Once the aggregates reach an optimal size, allow them to settle by gravity. Remove the medium.
Add differentiation media containing growth factors, and culture with a reduced bioreactor wheel speed. The growth factors promote neural differentiation within the aggregates.
Next, add fresh medium supplemented with growth factors and chemokines that induce the development of cerebellar progenitors.
Gradually, the cerebellar progenitors organize into distinct layers within the aggregates, forming early cerebellar organoids.
Finally, add a neuronal medium containing specific neurotrophic factors.
These factors induce cerebellar progenitor maturation into synaptically active functional cerebellar neurons, generating mature organoids.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

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 ...

JoVE Journal

Developmental Biology

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 ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors

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