Name: brain organoid generation from induced pluripotent stem cells in home-made mini bioreactors
Uploaded: 2021-12-11T15:00:00
Description: Watch this Scientific Journal Video about Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors at JoVE.com

This work was supported by grant 075-15-2019-1669 from the Ministry of Science and Higher Education of the Russian Federation (RT-PCR analysis) and by grant No. 19-15-00425 from the Russian Science Foundation (for all other work). The authors also thank Pavel Belikov for his help with the video editing. Figures in the manuscript were created with BioRender.com.

NOTE : Use sterile technique throughout the protocol, excluding steps 1.2 and 1.3. Warm all culture media and solutions to 37 &#176;C before applying to cells or organoids. Cultivate cells in a CO2 incubator at 37 &#176;C in 5% CO2 upon 80% humidity. The protocol scheme is shown in Figure 1.
1. Transforming Petri dishes into mini bioreactors
<ol>
	<li>Cut sterile 15 mL centrifuge tubes in rings of 7-8 mm in height; autoclav...

<ol>
	<li>Marchetto, M. C., Winner, B., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+in+neurodegenerative+and+neurodevelopmental+diseases.">Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases.</a> Human Molecular Genetics. 19, 71-76 (2010).</li><li>Lee, C. T., Bendriem, R. M., Wu, W. W., Shen, R. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Xiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+regionally+specified+hPSC+derived+organoids+models+human+brain+development+and+interneuron+migration.">Fusion of regionally specified hPSC derived organoids models human brain development and interneuron migration.</a> Cell Stem Cell. 21, 383-398 (2017).</li><li>Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+polarized+cerebellar+tissue+in+3D+culture+of+human+pluripotent+stem+cells.">Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells.</a> Cell Reports. 10 (4), 537-550 (2015).</li><li>Qian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+region+specific+organoids+using+mini+bioreactors+for+modeling+ZIKV+exposure.">Brain region specific organoids using mini bioreactors for modeling ZIKV exposure.</a> Cell. 165 (5), 1238-1254 (2016).</li><li>Qian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+brain+region+specific+organoids+using+a+miniaturized+spinning+bioreactor.">Generation of human brain region specific organoids using a miniaturized spinning bioreactor.</a> Nature Protocols. 13 (3), 565-580 (2018).</li><li>Jo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+like+organoids+from+human+pluripotent+stem+cells+contain+functional+dopaminergic+and+neuro+melanin+producing+neurons.">Midbrain like organoids from human pluripotent stem cells contain functional dopaminergic and neuro melanin producing neurons.</a> Cell Stem Cell. 19 (2), 248-257 (2016).</li><li>Sakaguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+hippocampal+neurons+from+self-organizing+human+embryonic+stem+cell+derived+dorsomedial+telencephalic+tissue.">Generation of functional hippocampal neurons from self-organizing human embryonic stem cell derived dorsomedial telencephalic tissue.</a> Nature Communication. 6 (1), 8896 (2015).</li><li>Di Lullo, E., Kriegstein, A. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+standardization.">Stem cell standardization.</a> Stem Cells Development. 20 (3), 375-377 (2011).</li><li>Jo, J., Xiao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+like+organoids+from+human+pluripotent+stem+cells+contain+functional+dopaminergic+and+neuro+melanin+producing+neurons.">Midbrain like organoids from human pluripotent stem cells contain functional dopaminergic and neuro melanin producing neurons.</a> Cell Stem Cell. 19 (2), 248-257 (2016).</li><li>Matsui, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six-month+cultured+cerebral+organoids+from+human+ES+cells+contain+matured+neural+cells.">Six-month cultured cerebral organoids from human ES cells contain matured neural cells.</a> Neuroscience Letters. 670, 75-82 (2018).</li><li>Trujillo, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+oscillatory+waves+emerging+from+cortical+organoids+model+early+human+brain+network+development.">Complex oscillatory waves emerging from cortical organoids model early human brain network development.</a> Cell Stem Cell. 25 (4), 558-569 (2019).</li><li>Eremeev, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necessity+Is+the+mother+of+invention&#8221;+or+inexpensive,+reliable,+and+reproducible+protocol+for+generating+organoids.">Necessity Is the mother of invention&#8221; or inexpensive, reliable, and reproducible protocol for generating organoids.</a> Biochemistry (Moscow). 84 (3), 321-328 (2019).</li><li>Qian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+brain+region&#8211;specific+organoids+using+a+miniaturized+spinning+bioreactor.">Generation of human brain region&#8211;specific organoids using a miniaturized spinning bioreactor.</a> Nature Protocols. 13, 565-580 (2018).</li><li>Matsui, T. K., Tsuru, Y., Hasegawa, K., Kuwako, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+of+human+brain+organoids.">Vascularization of human brain organoids.</a> Stem Cells. 39 (8), 1017-1024 (2021).</li><li>Hall, G. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned,+organoid-based+cartilaginous+implants+exhibit+zone+specific+functionality+forming+osteochondral-like+tissues+in+vivo.">Patterned, organoid-based cartilaginous implants exhibit zone specific functionality forming osteochondral-like tissues in vivo.</a> Biomaterials. 273, 120820 (2021).</li><li>Zachos, N. 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The described protocol has two crucial steps allowing the generation of high-quality organoids of uniform size. First, the organoids grow from spheroids which are near identical in cell number and cell maturity. Second, the home-made bioreactors provide each organoid a uniform environment, where organoids do not crowd or stick together.
The cell quality and state of cell maturation are essential to perform the protocol. It is critical to start neuronal differentiation at 75-90% confluence of i...

Human iPSC-derived models are widely used in the studies of neurodevelopmental and neurodegenerative disorders1. Over the past decade, 3D brain tissue models, so-called brain organoids, essentially complemented traditional 2D neuronal cultures2. The organoids recapitulate to some extent the 3D architecture of the embryonic brain and allow more precise modeling. Many protocols are published for the generation of organoids representing different brain regions: cerebral cortex3,4,5, cerebellum6, midbrain, forebrain, hypothalamus7,8,9, and hippocampus10. There have been multiple examples of using organoids to study human nervous system diseases11. Also, the organoids were implemented in drug discoveries12 and used in studies of infectious diseases, including SARS-Cov-213,14.
The brain organoids can reach up to several millimeters in diameter. So, the inner zone of the organoid may suffer from hypoxia or malnutrition and eventually become necrotic. Therefore, many protocols include special bioreactors8, shakers, or microfluidic systems15. These devices may require large volumes of expensive cell culture media. Also, the cost of such equipment is usually high. Some bioreactors consist of many mechanical parts that make them difficult to sterilize for reuse.
Most protocols suffer from the &#34;batch effect&#34;16, which generates significant variability among organoids obtained from the identical iPSCs. This variability hinders drug testing or preclinical studies requiring uniformity. The high yield of organoids enough to select organoids of uniform size may partially solve this problem.
The time factor is also a significant problem. Matsui et al. (2018) showed that brain organoids require at least six months to reach maturity17. Trujillo et al. (2019) also demonstrated that electrophysiological activity occurred in organoids only after six months of cultivation18. Due to the long organoid maturation time, the researchers often launch new differentiation before completing the previous one. Multiple parallel processes of differentiation require additional expenses, equipment, and laboratory space.
We have recently developed a mini bioreactor that mainly solves the problems mentioned above19. This home-made bioreactor consists of an ultra-low adhesion or untreated Petri dish with a plastic knob in the center. This plastic knob prevents crowding of organoids and their conglutination in the center of the Petri dish, which is caused by the rotation of the shaker. This paper describes how this inexpensive and simple home-made mini bioreactor allows generating high-quality brain organoids in large quantities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td>DMEM/F-12</td>
		</tr>
		<tr>
			<td>AggreWell400</td>
			<td>STEMCELL Technologies Inc</td>
			<td>34425</td>
			<td>24-well culture plate with microwells</td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>Neuronal supplement B</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>200 mM L-alanyl-L-glutamine</td>
		</tr>
		<tr>
			<td>Human BDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-2</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-839</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-290</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>Serum replacement</td>
		</tr>
		<tr>
			<td>mTESR1</td>
			<td>STEMCELL Technologies Inc</td>
			<td>85850</td>
			<td>Pliripotent stem cell medium</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Basal medium for neuronal cell maintenance</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>InvivoGen</td>
			<td>ant-mpt-1</td>
			<td>Antimicrobials</td>
		</tr>
		<tr>
			<td>Purmorphamine</td>
			<td>EMD Millipore</td>
			<td>540220</td>
			<td></td>
		</tr>
		<tr>
			<td>StemMACS Y27632</td>
			<td>Miltenyi Biotec</td>
			<td>130-106-538</td>
			<td>Y27632</td>
		</tr>
		<tr>
			<td>StemMACS Dorsomorphin</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-466</td>
			<td>Dorsomorphin</td>
		</tr>
		<tr>
			<td>StemMACS LDN-193189</td>
			<td>Miltenyi Biotec</td>
			<td>130-106-540</td>
			<td>LDN-193189</td>
		</tr>
		<tr>
			<td>StemMACS SB431542</td>
			<td>Miltenyi Biotec</td>
			<td>130-106-543</td>
			<td>SB431542</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Versen solution</td>
			<td>Gibco</td>
			<td>15040066</td>
			<td>0.48 mM EDTA in PBS</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350010</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The protocol scheme is shown in Figure 1. The protocol included five media in which iPSCs differentiated into brain organoids during at least one month. The differentiation was started then iPSCs reached the 75-90% confluence (Figure 2A,B). The first signs of differentiation towards neurons were observed on days 10-11 of iPSC cultivation in medium A when cells began to cluster into &#34;rosettes&#34; (Figure 2C). At...

Watch this Scientific Journal Video about Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors at JoVE.com

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

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 ...

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