generation of spheroids from human pluripotent stem cells

Generation of Spheroids from Human Pluripotent Stem Cells

To begin, plate the hPSC colonies on an hESC-qualified basement membrane matrix from one well of a 6-well plate at 60% confluency into three wells to achieve 20% to 30% density, and then, proceed with induction of 2D neuroectodermal colonies.
Add fresh N2 media supplemented with dual SMAD inhibitors daily to each well for the next 3 days. For generating 3D neuroectodermal spheroids from induced 2D neuroectodermal colonies, remove 2 milliliters of N2 medium from the 6-well plate, and wash one time with HBSS to ensure that all of the N2 medium is removed.
Add 1 milliliter of dispase to each colony-containing well. Incubate the plate for 20 to 25 minutes at 37 degrees Celsius, and check for colony detachment regularly. At the end of the incubation, to stop the activity of the dispase enzyme, add 1 milliliter of N2 medium to the well. Using a wide-bore P1000 pipette tip or a modified P1000 pipette tip cup with sterile scissors, transfer the colonies into a 15-milliliter tube, and allow the colony clumps to sink to the bottom of the tube with gravity.
Then, with a standard P1000 pipette tip, carefully remove the supernatant. Replace it with 1 milliliter of fresh N2 medium, and repeat washing thrice to ensure complete removal of dispase. After resuspending the cell clumps and N2 medium, transfer the cell suspension to one well of a 6-well plate, and add 40 nanograms per milliliter of bFGF.

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1. Cortical brain organoid generation (Figure 1) 
NOTE: All the steps in this section of the protocol will occur in a Class 2 biosafety hood unless stated otherwise.
<ol>
	<li>Induction of 2D neuroectodermal colonies from human pluripotent stem cells (hPSCs) 2D culture (Days -1 to 3)
	<ol>
		<li>Before induction, plate the hPSC colonies on a human embryonic stem cell (hESC) qualified basement membrane matrix in a 6-well plate at 20%-30% density. Achieve this density by passaging hPSC colonies from one well of a 6-well plate at 60% confluency into three wells of a 6-well plate.</li>
		<li>For basement membrane matrix coating, dilute the basement membrane matrix at a ratio of 1:50 in a plain basal medium. Evenly deposit 1 mL/well of a 6-well plate, incubate for 1 hour at room temperature (RT), and then aspirate.</li>
		<li>Maintain hPSC colonies for 1 day in 2 mL of a serum-free cell culture medium prior to differentiation.</li>
		<li>On the day of hPSC differentiation, inspect the hPSC colonies using brightfield microscopy at 4x to 10x magnification to ensure healthy colonies with no detectable differentiation. 
		NOTE: Healthy hPSCs will form tight edge colonies with cells that have a large nucleus, very small cytoplasm, and prominent nucleoli. Differentiated iPSC colonies will exhibit clear morphological differences to that of the described hPSC colonies above, particularly around the outer edges of the colonies or at the center.</li>
		<li>Add reagents listed in Table 1 to make up the N2 medium required for differentiation. Bring this medium to RT before use.</li>
		<li>Once at RT, aspirate the serum-free cell culture medium from each well of the 6-well plate and replace it with 2 mL of N2 medium gently added with a 5 mL serological pipette.</li>
		<li>Add the dual Suppressor of Mothers against Decapentaplegic (SMAD) inhibitors, SB-431542 (10 &#181;M) and LDN 193189 (100 nM). 
		NOTE: The SMAD inhibitors can be added to the N2 medium after the medium has been placed in each well or to the required amount of N2 medium prior to replacing the serum-free cell culture medium. Inhibitors can be evenly incorporated into the medium by gently swirling the plate or inverting the tube containing the medium and inhibitors 3-4 times.</li>
		<li>Add fresh N2 media supplemented with SB-431542 (10 &#181;M) and LDN 193189 (100 nM) daily to each well for the next 2 days. 
		NOTE: Fresh N2 medium is added to reduce the prolonged exposure of cells to the Dimethyl sulfoxide (DMSO) that is used to dissolve SB-431542 and LDN 193189 compounds and prevent cytotoxicity.</li>
	</ol>
	</li>
	<li>Generation of 3D neuroectodermal spheroids from induced 2D neuroectodermal colonies (Days 3 to 7)
	<ol>
		<li>Lift the induced neuroectodermal colonies using dispase following steps 1.2.2-1.2.8.</li>
		<li>First, remove 2 mL of N2 medium from the 6-well plate, and wash 1x with HBSS to ensure that all of the N2 medium is removed. 
		NOTE: N2 medium can interfere with the enzyme activity of dispase, preventing adequate detachment of neuroectodermal colonies from the well.</li>
		<li>Add 1 mL of 2.4 unit/mL dispase to each colony-containing well.</li>
		<li>Incubate the well for 20-25 min (maximum 30 min) at 37 &#176;C. Check for colony detachment regularly. 
		NOTE: Small colonies may detach within 20 minutes. Any colonies that remain stuck down after 30 min should be ignored.</li>
		<li>After incubation, add 1 mL of N2 medium to the well to stop the activity of the dispase enzyme and transfer the colonies into a 15 mL tube using a wide-bore P1000 pipette tip or a modified P1000 pipette tip cut with sterile scissors (making it a wide-bore P1000 tip).</li>
		<li>Allow the colony clumps to sink to the bottom of the tube with gravity. 
		NOTE: This process will take approximately 1 minute.</li>
		<li>Once the clumps have sunk, carefully remove the supernatant with a standard P1000 pipette tip and replace it with 1 mL of fresh N2 medium. Repeat this washing step three times to ensure complete removal of dispase. 
		NOTE: Any remaining dispase will prevent a uniform formation of neuroectodermal spheroids and induce cell death.</li>
		<li>After washing, resuspend the cell clumps in 3 mL of N2 medium, transfer to one well of a 6-well plate, and add 40 ng/mL of basic fibroblast growth factor (bFGF). 
		NOTE: If a high number of neuroectodermal colonies were detached, these colonies could be plated across two or more wells of a 6-well plate to prevent spheroids fusion. 24 h after dispase detachment of the colonies, check whether the spheroids have formed.</li>
		<li>Maintain the spheroids in the same media for the next 3-4 days but add fresh bFGF (40 ng/mL) to each well daily to promote neuroectodermal cell proliferation, self-organizing, and induce and expand neuroepithelia. 
		NOTE: If spheroids have been plated at a higher density, the media is likely to turn yellow and require replacing every 2 days with fresh bFGF (40 ng/mL). However, this is not recommended, and instead, a lower number of spheroids should be maintained in each well to avoid this issue. Spheroids can be embedded in the basement membrane matrix after 3 days if neuroepithelia are evident. If neuroepithelia are not evident or do not look strong, then maintain the spheroids for another day and check again.</li>
	</ol>
	</li>
</ol>
Table 1: N2 Medium. The table lists the reagents required to prepare the N2 medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media components</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>DMEM Nutrient mix F12 10x 500 mL (DMEM/F-12)</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement 5 mL (100x)</td>
			<td>Supplmented at 1%</td>
		</tr>
		<tr>
			<td>B 27 Supplement 10 mL</td>
			<td>Supplemented at 2%</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Supplemented at 1%</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Supplemented at 1%</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol 50 mL(1000x )</td>
			<td>Supplemented at 0.1%</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% Formaldehyde (W/V) Methanol-free</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>4% of PFA are diluted in 1x PBS</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol 50 mL(1000x)</td>
			<td>Life Technologies Australia (TFS)</td>
			<td>21985023</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>B 27 Supplement 10 mL</td>
			<td>Life Technologies Australia (TFS)</td>
			<td>17504044</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>CKX53 microscope with SC50 camera</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar 6 well cell culture plates</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>CLS3516-50EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>17105041</td>
			<td>Powder is dissolve in HBSS, filtered through 0.22 &#181;m filter, aliquote at 10 mL and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>DMEM Nutrient Mix F12 10x 500 mL (DMEM/F-12)</td>
			<td>Thermofisher</td>
			<td>11320082</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>DMSO Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>D1408-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Matrigel hESC-qualified Matrix</td>
			<td>In Vitro Technologies Pty Ltd</td>
			<td>FAL354277</td>
			<td>Make aliquotes of 100 &#181;L and stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement 100x</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>Human induced pluripotent stem cells (EU79)</td>
			<td></td>
			<td></td>
			<td>In-house reporogrammed from skin fibroblast</td>
		</tr>
		<tr>
			<td>Human induced pluripotent stem cells (G22)</td>
			<td>Genea Biocells</td>
			<td></td>
			<td>Obtained from Genea Biocells (San Diego, United States)</td>
		</tr>
		<tr>
			<td>Human induced pluripotent stem cells (WTC)</td>
			<td></td>
			<td></td>
			<td>Gift from Professor Bruce Conklin</td>
		</tr>
		<tr>
			<td>InSolution TGF-&#914; RI Kinase Inhibitor VI, SB431542</td>
			<td>Merck</td>
			<td>US1616464-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Solution Human Recombinant</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>I9278</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>LDN193189 Dihydrochloride</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>SML0559-5MG</td>
			<td>Used during differentiation</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>STEMCELL TECHNOLOGIES</td>
			<td>100-0276</td>
			<td>Used to maintain hiPSC colonies prior to differentiation with NM media</td>
		</tr>
		<tr>
			<td>N2 Supplement 5 mL (100x)</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>17502048</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Used in NM and DM media</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF basic</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-01M</td>
			<td>Aliquots are made at 20 &#181;g/mL and stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td>Used during differentiation</td>
		</tr>
		<tr>
			<td>Ultra-Low attachment multiwell plates, 24-well plate, polystyrene</td>
			<td>Sigma Aldrich Pty Ltd</td>
			<td>CLS3473-24EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Shaker, M. R., et al. <a href="https://app.jove.com/v/63714">Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.</a> J. Vis. Exp. (2022).
This video demonstrates the transformation of human pluripotent stem cells (hPSCs) into spherical brain organoids. By using specific inhibitors, the hPSCs are guided to differentiate towards a neuronal lineage, forming neuroectodermal colonies. Special growth factors are then introduced, which encourage the self-organization and proliferation of the colonies, ultimately leading to the formation of spherical brain organoids.

<img alt="Figure 1" src="/files/ftp_upload/22431/22431_Figure1.jpg" /> 
Figure 1: Schematic diagram for generating reproducible cortical brain organoids. Schematic workflow of the experimental procedure for the generation of cortical brain organoids from hPSCs maintained in the feeder-free medium. The workflow provides an overview of six steps involved in differentiating the 2D hPSCs into 3D patterned cortical plate human tissues in organoids.

1. Cortical brain organoid generation (Figure 1) 
NOTE: All the steps in this section of the protocol will occur in a Class 2 biosafety hood unless stated ...

Begin with colonies of human pluripotent stem cells or hPSCs attached to a suitable substrate in a culture medium.
Remove the medium. Add a neuronal medium containing specific inhibitors and incubate.
These inhibitors direct the maturation of hPSCs toward a neuronal lineage, forming neuroectodermal colonies. These are precursors to small, spherical structures called spheroids.
Remove the medium and wash with a salt buffer.
Incubate with a digestive enzyme to detach the colonies from the plate.
Add the neuronal medium to stop the enzymatic activity.
Collect the colonies and transfer them to a tube.
Allow them to settle, and then discard the supernatant.
Resuspend the colonies in a neuronal medium containing a growth factor.
Transfer the colonies to a multi-well plate and incubate.
This growth factor facilitates the self-organization and multiplication of neuroectodermal cells, leading them to form a spheroid.

20 Views. Получение сфероидов из плюрипотентных стволовых клеток человека

Video: Получение сфероидов из плюрипотентных стволовых клеток человека - Experiment

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.

Генерация IPSC, полученные из органоидов человеческого мозга Модели раннего развития нервной системы Расстройства

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.

Генерация органоидов мозга из индуцированных плюрипотентных стволовых клеток в самодельных мини-биореакторах

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.

Трехмерное моторное нервное органоидное поколение

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

Generation of Spheroids from Human Pluripotent Stem Cells

ТРАНСКРИПТ

Generation of Spheroids from Human Pluripotent Stem Cells