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

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

On day 1, with day 0 being the day of seeding the bioreactor, place the bioreactor and the base unit in a sterile flow hood. Then, use a serological pipette to collect a 1-milliliter sample of the cell suspension. Plate the cell suspension in an ultra-low attachment 24-well plate. Use a microscope to confirm that iPSC-derived aggregates have formed. If so, capture images of the aggregates at 40x or 100x.
Continue culturing the iPSCs in the bioreactor until the average diameter of the aggregates is 100 micrometers. Then, replace 80% of the medium with fresh mTesR1 without ROCK inhibitor. When the aggregates reach 200 to 250 micrometers in diameter, let all the organoids settle at the bottom of the bioreactor. Then, replace all the spent medium with gfCDM differentiation medium.
Place the base unit and the bioreactor in the incubator and decrease agitation to 25 RPM.

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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&#215; PBS per well.</li>
	<li>Add 1 mL of the cell detachment medium (see Table of Materials) to each well of a 6-well plate and incubate at 37 &#176;C for 7 min until cells detach easily from the wells with gentle shaking.</li>
	<li>Pipette the cell detachment medium up and down with a P1000 micropipette until the cells detach and dissociate into single cells. Add 2 mL of complete cell culture medium to each well to inactivate enzymatic digestion and pipette the cells gently into a sterile conical tube.</li>
	<li>Centrifuge at 210 &#215; g for 3 min and remove the supernatant.</li>
	<li>Resuspend the cell pellet in culture medium (i.e., mTeSR1 supplemented with 10 &#181;M of ROCKi) and count the iPSCs with a hemocytometer using trypan blue dye.</li>
	<li>Seed 15 &#215; 106 single cells in the bioreactor (maximum volume of 100 mL) with 60 mL of mTeSR1 supplemented with 10 &#181;M of ROCKi at a final cell density of 250,000 cells/mL.</li>
	<li>Insert the vessel containing the iPSCs in the universal base unit placed in the incubator at 37 &#176;C, 95% humidity, and 5% CO2. 
	NOTE: The bioreactor stirring is maintained for 24 h by setting the universal base unit control to 27 rpm to promote iPSC aggregation.</li>
</ol>
2. Differentiation and maturation of human iPSC-derived aggregates in cerebellar organoids
<ol>
	<li>Define the day of single cell seeding as day 0.</li>
	<li>On day 1, collect 1 mL of the iPSC aggregates sample using a serological pipette. Maintain the bioreactor under agitation as before by placing the universal base unit with the bioreactor containing the aggregates in a sterile flow prior to collecting the sample. Plate the cell suspension in an ultra-low attachment 24-well plate. Check that iPSC-derived aggregates are formed.</li>
	<li>Acquire images with an optical microscope using a total magnification of 40x or 100x to measure aggregate diameter.</li>
	<li>Measure the area of the aggregates in each image using FIJI software.
	<ol>
		<li>Select &#34;Analyze | Set Measurements&#34; from the menu bar and click on &#34;Area&#34; and &#34;OK&#34;.</li>
		<li>Select &#34;File | Open&#34; from the menu bar to open a stored image file. Select the line selection tool presented in the toolbar and create a straight line over the scale bar presented in the image. Select &#34;Analyze | Set scale&#34; from the menu bar.</li>
		<li>In &#34;Known distance,&#34; add the expanse of the image&#39;s scale bar in &#181;m. Define the &#34;Unit of length&#34; as &#181;m. Click on &#34;Global&#34; to maintain the settings and &#34;OK&#34;. Select Oval Selection in the toolbar.</li>
		<li>For each aggregate, delineate the area with the oval tool. Select &#34;Analyze | Measure&#34;. Calculate their diameter based on measured area, considering that aggregates are approximately spherical using 
		<img alt="Equation 1" src="/files/ftp_upload/22386/22386eq01.jpg" style="margin: auto;" /> 
		with A as the area of the aggregate.</li>
	</ol>
	</li>
	<li>&#160;When the average diameter of the aggregates is 100 &#181;m, replace 80% of the spent medium with fresh mTeSR1 without ROCKi. When aggregates reach 200&#8211;250 &#956;m in diameter, replace all the spent medium with gfCDM (Table 1), letting the organoids settle at the bottom of the bioreactor. 
	NOTE: If the average aggregate diameter exceeds 350 &#956;m, do not start the differentiation protocol. Repeat the seeding of single cells. Generally, it takes around 1 day for the aggregate to reach an average diameter of 100 &#181;m.</li>
	<li>Insert the bioreactor containing the aggregates in the universal base unit placed in the incubator at 37 &#176;C, 95% humidity, and 5% CO2.</li>
	<li>Decrease the bioreactor agitation to 25 rpm.</li>
	<li>On day 2, repeat steps 2.2, 2.3, and 2.4 to evaluate the aggregate diameter. Add 30 &#956;L of FGF2 (final concentration, 50 ng/mL) and 60 &#956;L of SB431542 (final concentration, 10 &#956;M) to 60 mL of gfCDM differentiation medium (Table 1). Replace all spent medium from the bioreactor with the supplemented gfCDM. Repeat step 2.6. 
	NOTE: SB431542 is crucial to inhibit mesendodermal differentiation, inducing neural differentiation. FGF2 is used to promote the caudalization of the neuroepithelial tissue.</li>
	<li>On day 5, repeat steps 2.2, 2.3, 2.4, and 2.8. 
	NOTE: Aggregate size should increase during the differentiation protocol. However, the diameter is only critical when the differentiation starts because this parameter could influence the efficacy of differentiation.</li>
	<li>On day 7, repeat steps 2.2, 2.3, and 2.4. Dilute FGF2 and SB431542 to 2/3: Add 20 &#956;L of FGF2 and 40 &#956;L of SB431542 to 60 mL of gfCDM differentiation medium. Replace all spent medium from the bioreactor with supplemented gfCDM. Repeat step 2.6 and increase bioreactor agitation to 30 rpm.</li>
	<li>On day 14, repeat steps 2.2, 2.3, and 2.4. Add 60 &#956;L of FGF19 (final concentration, 100 ng/mL) to 60 mL of gfCDM differentiation medium. Replace all spent medium from the bioreactor with gfCDM supplemented with FGF19. Repeat step 2.6. 
	NOTE: FGF19 is used to promote polarization of mid-hindbrain structures.</li>
	<li>On day 18, repeat steps 2.2, 2.3, 2.4, and 2.11.</li>
	<li>On day 21, repeat steps 2.2, 2.3, and 2.4. Replace all spent medium from the bioreactor with complete neurobasal medium (Table 1). Repeat step 2.6. 
	NOTE: Neurobasal medium is a basal medium used to maintain the neuronal cell population within the organoid.</li>
	<li>On day 28, repeat steps 2.2, 2.3, and 2.4. Add 180 &#956;L of SDF1 (final concentration, 300 ng/mL) to 60 mL of complete neurobasal medium. Replace all spent medium from the bioreactor with complete neurobasal medium supplemented with SDF1. Repeat step 2.6. 
	NOTE: SDF1 is used to facilitate the organization of distinct cell layers.</li>
	<li>On day 35, repeat steps 2.2, 2.3, and 2.4. Replace all spent medium from the bioreactor with complete BrainPhys medium (Table 1). Repeat step 2.6. 
	NOTE: BrainPhys is a neuronal medium that supports synaptically active neurons.</li>
	<li>Replace 1/3 of the total volume every 3 days with complete BrainPhys medium until day 90 of differentiation.</li>
</ol>
Table 1: Stock solutions and media preparation. Listed are all the components and volumes used to prepare media for the iPSCs maintenance and differentiation protocol, as well as stock solutions of growth factors and small molecules. For stock solutions, all stock concentrations and protocols for reconstitution are listed.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="4">Media preparation</td>
			<td>mTeSR1 
			Final volume: 500 mL</td>
			<td>1. Thaw mTeSR1 5&#215; supplement at room temperature (RT) or at 4 &#176;C overnight and mix with basal medium 
			2. Store complete mTeSR1 medium at 4 &#176;C for up to 2 weeks or prepare 40 mL aliquots and store at -20 &#176;C 
			3. Pre-warm complete mTeSR1 at RT before use</td>
		</tr>
		<tr>
			<td>gfCDM (growth factor-free chemically defined medium) 
			Final volume: 60 mL</td>
			<td>30 mL Ham&#39;s F12 30 mL IMDM 600 &#181;L chemically defined lipid concentrate (1 % v/v) 2.4 &#181;L monothioglycerol (450 &#956;M) 30 &#181;L apo-transferrin (stock solution at 30 mg/mL in water, final concentration: 15 &#956;g/mL) 300 mg crystallization-purified BSA (5 mg/mL) 42 &#181;L insulin (stock concentration at 10 mg/mL, final concentration: 7 &#181;g/mL) 300 &#181;L P/S (0.5% v/v, 50 U/ml penicillin/50 &#956;g/ml streptomycin)</td>
		</tr>
		<tr>
			<td>Neurobasal 
			Final volume: 60 mL</td>
			<td>60 mL of Neurobasal medium 600 &#181;L N2 supplement 600 &#181;L Glutamax I 300 &#181;L P/S (0.5 % v/v).</td>
		</tr>
		<tr>
			<td>Complete BrainPhys 
			Final volume: 60 mL</td>
			<td>60mL of BrainPhys 1.2 mL NeuroCult SM1 Neuronal Supplement 600 &#181;L N2 Supplement 12 &#181;L BDNF (final concentration: 20 ng/mL) 12 &#181;L GDNF (final concentration: 20 ng/mL) 300 &#181;L Dibutyryl-cAMP (stock concentration: 100 mg/mL in water, final concentration: 1 mM) 42 &#181;L ascorbic acid (stock concentration: 50 &#181;g/mL in water, final concentration: 200 nM)</td>
		</tr>
		<tr>
			<td rowspan="8">Stock solutions of growth factors and small molecules</td>
			<td>Basic fibroblast growth factor (bFGF/FGF2) 
			Stock concentration: 100 &#181;g/mL</td>
			<td>1. Reconstitute in 5 mM Tris, pH 7.6, at a concentration of 10 mg/mL 
			2. Dilute with 0.1 % BSA in PBS (v/v) to a final stock concentration of 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Stromal cell-derived factor 1 (SDF1) 
			Stock concentration: 100 &#181;g/mL</td>
			<td rowspan="3">1. Reconstitute in water at a concentration of 10 mg/mL 
			2. Dilute with 0.1 % BSA (v/ v) in PBS to a final stock concentration of 100 &#181;g/mL.</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF) 
			Stock concentration: 100 &#181;g/mL&#60;</td>
		</tr>
		<tr>
			<td>Glial cell-derived neurotrophic factor (GDNF) 
			Stock concentration: 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 19 (FGF19) 
			Stock concentration: 100 &#181;g/mL</td>
			<td>1. Reconstitute in 5 mM sodium phosphate, pH 7.4, at a concentration of 10 mg/mL 
			2. Dilute with 0.1 % BSA in PBS (v/v) to a final stock concentration of 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632 
			Stock concentration: 10mM</td>
			<td rowspan="2">Reconstitute in DMSO at a concentration of 10 mM.</td>
		</tr>
		<tr>
			<td>SB431542 
			Stock concentration: 10mM</td>
		</tr>
		<tr>
			<td>Insulin 
			Stock concentration: 10 mg/mL</td>
			<td>1. Reconstitute 10 mg of insulin in 300 &#181;L of 10 mM NaOH 
			2. Carefully add 1 M NaOH until the solution becomes clear-transparent 
			3.Fill to 1 mL with sterile water</td>
		</tr>
	</tbody>
</table>
Table 2: Solutions for preparation of organoids for cryosectioning and immunostaining. Listed are all the components and volumes used to prepare the solutions used in the preparation of organoids for cryosectioning and immunostaining.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gelatin/Sucrose 
			Final concentration: 7.5%/15% w/w</td>
			<td>1. Weigh 15 g of sucrose and 7.5 g of gelatin in a sterile Schott Glass Bottle and mix well 
			2. Pre-warm the PBS 1&#215; at 65 &#176;C 
			3. Add pre-warmed PBS 1&#215; to a final weight of 100 g and mix well 
			4. Place the Schott Glass Bottle in a heating plate at 65 &#176;C and shake until the gelatin melts 
			5. Incubate at 37 &#176;C until the solution stabilizes</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BrainPhys Neuronal Medium N2-A &#38; SM1 kit</td>
			<td>5793 - 500mL</td>
			<td>Stem cell tecnhnologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically defined lipid concentrate</td>
			<td>11905031</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallization-purified BSA&#160;</td>
			<td>5470</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>32500-035</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>A3840001</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>G9391</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax I</td>
			<td>10566-016</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutyryl cAMP&#160;</td>
			<td>SC- 201567B -500mg&#160;</td>
			<td>Frilabo</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>21765029</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Episomal iPSC Line</td>
			<td>A18945</td>
			<td>ThermoFisher</td>
			<td>iPSC6.2</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>12440046</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>91077C</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>iPS DF6-9-9T.B</td>
			<td></td>
			<td>WiCell</td>
			<td></td>
		</tr>
		<tr>
			<td>Iso-pentane</td>
			<td>PHR1661-2ML</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>A-92902</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>354230</td>
			<td>Corning</td>
			<td>basement membrane matrix</td>
		</tr>
		<tr>
			<td>Monothioglycerol</td>
			<td>M6154</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>85850 -500ml</td>
			<td>Stem cell technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>17502048</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>12348017</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>158127</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-0.1 Single-Use Vessel</td>
			<td>SKU: IA-0.1-D-001</td>
			<td>PBS Biotech</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-MINI MagDrive Base Unit</td>
			<td>SKU: IA-UNI-B-501</td>
			<td>PBS Biotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human BDNF</td>
			<td>450-02</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human bFGF/FGF2</td>
			<td>100-18B</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human FGF19</td>
			<td>100-32</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human GDNF</td>
			<td>450-10</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human SDF1</td>
			<td>300-28A</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor</td>
			<td>Y-27632 72302</td>
			<td>Stem cell technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>S4317</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>S7903</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>25608-930</td>
			<td>VWR</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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.

30 조회수. Single-Use Bioreactors를 사용하여 유도 만능 줄기 세포에서 성숙한 소뇌 오가노이드 생성

비디오: Single-Use Bioreactors를 사용하여 유도 만능 줄기 세포에서 성숙한 소뇌 오가노이드 생성 - 실험

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.

IPSC 파생 된 인간의 두뇌 Organoids의 세대는 초기 신경 발달 장애를 모델링하기

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

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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.

집에서 만든 미니 생물 반응기에서 유도 만능 줄기 세포에서 뇌 오르간성 생성

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.

3차원 모터 신경 오르간성 생성

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