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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+brain+Organoids+derived+from+pluripotent+stem+cells:+promising+experimental+models+for+brain+development+and+neurodegenerative+disorders.">3D brain Organoids derived from pluripotent stem cells: promising experimental models for brain development and neurodegenerative disorders.</a> Journal of Biomedical Science. 24 (1), 1-12 (2017).</li><li>Kadoshima, T., 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=Self-organization+of+axial+polarity,+inside+out+layer+pattern,+and+species-specific+progenitor+dynamics+in+human+ES+cell-derived+neocortex.">Self-organization of axial polarity, inside out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.</a> Proceedings of the National Academy of Sciences U.S.A. 110 (50), 20284 (2013).</li><li>Lancaster, M. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+brain+organoids+to+investigate+neural+development+and+disease.">The use of brain organoids to investigate neural development and disease.</a> Nature Reviews Neuroscience. 18 (10), 573-584 (2017).</li><li>Chen, K. G., 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=Pluripotent+stem+cell+platforms+for+drug+discovery.">Pluripotent stem cell platforms for drug discovery.</a> Trends in Molecular Medicine. 24 (9), 805-820 (2018).</li><li>Dang, 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=Zika+virus+depletes+neural+progenitors+in+human+cerebral+organoids+through+activation+of+the+innate+immune+receptor+TLR3.">Zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3.</a> Cell Stem Cell. 19 (2), 258-265 (2016).</li><li>Tiwari, S. K., Wang, S., Smith, D., Carlin, A. F., Rana, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+tissue-specific+SARS-CoV-2+infection+and+host+responses+using+human+stem+cell-derived+lung+and+cerebral+organoids.">Revealing tissue-specific SARS-CoV-2 infection and host responses using human stem cell-derived lung and cerebral organoids.</a> Stem Cell Reports. 16 (3), 437-445 (2021).</li><li>Ao, Z., 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=One-stop+microfluidic+assembly+of+human+brain+organoids+to+model+prenatal+cannabis+exposure.">One-stop microfluidic assembly of human brain organoids to model prenatal cannabis exposure.</a> Analytical Chemistry. 92 (6), 4630-4638 (2020).</li><li>Di Nardo, P., Parker, G. 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. C., 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=Human+enteroids/colonoids+and+intestinal+organoids+functionally+recapitulate+normal+intestinal+physiology+and+pathophysiology.">Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology.</a> Journal of Biological Chemistry. 291, 3759-3766 (2016).</li><li>Eremeev, 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&#8212;challenges+to+establish+a+brain+prototype.">Cerebral organoids&#8212;challenges to establish a brain prototype.</a> Cells. 10 (7), 1790 (2021).</li><li>Kadoshima, T., 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=Self-organization+of+axial+polarity,+inside-out+layer+pattern,+and+species-specific+progenitor+dynamics+in+human+ES+Cell-derived+neocortex.">Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES Cell-derived neocortex.</a> Proceedings of the National Academy of Sciences U. S. A. 110, 20284-20289 (2013).</li></ol>

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 ...

To date, the cerebral organoids differentiated from pluripotent stem cells is an vitro 3D model closest to the native human brain tissue. Additionally, cerebral organoids are equally suitable for modeling various central nervous system pathologies, testing pharmacologically-active substances, and also for use in regenerative medicine. Meanwhile, this technology is still at the initial stage of development.The protocol can be divided into two groups depending on the method of obtaining aggregates and the further cultivation with differentiation. The first includes protocol and schemes of differentiation, different in the cultivation time and differentiation inducers with subsequent maturation of organoid in the shaker in special low-adhesion devices like market tubes or plates. Another group includes using a special bioreactors.Analysis of various protocols has shown that organoids should be cultivated under conditions with circulating nutrient medium. However, the absence of simple and inexpensive systems for obtaining organoid with a standard size and morphology require the development of such devices for scaling the process. In this work, we describe our method for obtaining and testing simple and inexpensive mini bioreactors that make it possible to obtain large quantities of high-quality organoids.Cut the sterile 15 milliliters centrifuge tubes into the rings, 7 or 8 millimeters in height. Autoclave the rings. Break low-aggregations on treated or microbiological Petri dishes into crumbs.Dissolve about 1 grams of plastic crumbs in 10 milliliters of chloroform overnight to prepare a liquid plastic. Make a plastic knob in the center of a sterile ultra low-adhesion 6 centimeters Petri dish. There are two equally suitable ways.The first, put the autoclave in a plastic ring on a center and apply half milliliters of liquid plastic to the inside of the ring. The second, without any plastic rings drop half milliliters of liquid plastic on the center of the Petri dish. Leave the dishes open for 2 or 3 hours in a laminar flow hood until complete dryness.Cultivate induced pluripotent stem cells in the medium for pluripotent stem cells, up to 75 or 90%confluence in the 35 millimeters Petri dishes, pre-coated with a matrix dissolved in cold Dulbecco Modified Eagle Medium, and Fisher-12 medium. Prepare medium A plus serum replacement. Cultivate induced pluripotent stem cells for 1 day in the medium with serum replacement.Change medium for pluripotent stem cells to a serum replacement medium at the day zero of differentiation. Prepare medium A.Change the medium to medium A at day 2 of differentiation. Cultivate cells in medium A for 2 weeks, refreshing medium in Petri dishes every 2 day.At day 14, start the formation of spheroid using a special 24-well culture plate which contains approximately 1, 200 microwells in each well. Prepare a 24-well culture plate with microwells. Add to each well 1 milliliter of medium A, centrifuge briefly at 1, 300g for 5 minutes in a swinging-bucket rotor fitted with plate holder.Control under the microscope that there are no bubbles in microwells. Prepare the medium B.Remove the medium from the Petri dish. For the cell detachment, treat the cells with 1.5 milliliters EDTA solution prepared on PBS.Control the cell detachment under the microscope. Harvest the cell into 15 milliliter tube. Add 5 milliliter mixed Dulbecco and Fisher medium in the tube towards the cells.Centrifuge at 200g for 5 minutes. Remove the supernatant and re-suspend cells in 2 milliliter of medium B.Transfer the cells suspension containing 1 billion cells into each well of a 24-well plate with microwells. Gently pipette cells up to and down several times and centrifuge briefly 100g for 1 minute to capture cell in microwells.Control under the microscope that the cell are evenly distributed in microwells. Incubate the plate overnight for cell aggregation in spheroids. The next morning, day 15, check under the microscopes the quality of spheroids, which are transparent and smooth if healthy.Carefully collect the spheroids from each well into a 15 milliliter tube, leave the spheroids to precipitate by gravity for 2 and 3 minutes, and then remove the supernatant. Add to the spheroids 2 milliliters of the matrix, thawed and ex tempore on ice. Mix gently by pipetting and incubate at room temperature for 30 minutes.To wash excess of the matrix, add to the tube 8 milliliters of medium B.Pipette gently, then centrifuge the tube for 1 minutes at 100g. Remove the supernatant. Add to the tube 20 milliliters of medium B, pipette gently, then split the spheroid suspension between mini bioreactors.Then place the mini bioreactors into a 15 centimeter Petri dish, which prevents the evaporation of water and contamination. Put the Petri dish with mini bioreactors on a orbital shaker. Cultivate the organoids at rotation rate 70, 75rpm.On day 16, prepare medium C.Transfer organoid into 50 milliliter tube. For 5 minutes, let them fall to the bottom. Aspirate the supernatant and add 5 milliliters of the medium C.Rejoin the organoids in the mini bioreactors.Cultivate spheroids in medium C for 2 weeks, refreshing the medium every 2 days. During these 2 weeks, select about 100 spheroids per mini bioreactors for the following cultivation. On day 30, prepare medium D.Change cultivation medium to medium D, which is a maturation medium.Refresh cultivation medium every 2 or 3 days for 3 weeks. Then use the medium D dissolved in F and GDNF. After 1 month of consecutive cultivation in medium A and B, organoids standardized in size and morphology are obtained.As it grows, they need to change the medium more often, up to 4 times a week also increases. After 2 months of cultivation, they reach 4 and 5 millimeter in diameter. And after another month, 5 and 6 millimeter and the growth stops.The figure shows the appearance of organoid enzyme, clear sections painted with hematoxylin and eosin. Immunohistochemical analysis for 1 month shows the presence of SOX2 positive clusters of progenitor cells, including inside central part. In the peripheral of the organoids, glial fibrillary acidic protein, microtubule-associated protein 2, and thiazine hydrolase positive.Cells are concentrated, which corresponds to mature form of neurons. In some cases, clusters of whites on necrotic area are formed in the central part. This indicates the limitation of this protocol for obtaining larger organoids.Most likely, the limiting factor of growth is the rate of diffusion of nutrient and oxygen. The various media and cell type of spheroids and testing our system for some kinds of organoids. Lots of brain and retina, combined organoids from liver carcinoma cells and mesenchymal stem cells, combined organoids from capital blast mesenchymal stem calls and the hematopoietic stem cells differentiation from induced pluripotent stem cells and intestine organoids.Was it variable in the initial composition of cells? Differentiation factors, culture, and medium, extracellular matrix, and possibly also the gas mixture composition, the rate of the platform rotation, and other parameters. It will be possible to obtain organoid with the same shape, size, or morphology model in various organs and tissues.The use of mini bioreactors proposed into this work.

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JoVE Journal

Developmental Biology

4.9K 조회수.  Federal Medical Biological Agency. 현재까지, 다능성 줄기 세포에서 분화된 대뇌 오르간노이드는 토착 인간 뇌 조직에 가장 가까운 시험관 3D 모델이다. 또한 대뇌 오르가노이드는 다양한 중추 신경계 병리학을 모델링하고, 약리활성 물질을 테스트하며, 재생 의학에도 사용하기에도 똑같이 적합합니다. 한편,이 기술은 여전히 개발의 초기 단계에있다.프로토콜은 골재를 획득하는 방법과 분화와 함께 추가...