This work was supported by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT), Portugal (UIDB/04565/2020 through Programa Operacional Regional de Lisboa 2020, Project N. 007317, PD/BD/105773/2014 to T.P.S and PD/BD/128376/2017 to D.E.S.N.), projects co-funded by FEDER (POR Lisboa 2020&#8212;Programa Operacional Regional de Lisboa PORTUGAL 2020) and FCT through grant PAC-PRECISE LISBOA-01-0145-FEDER-016394 and CEREBEX Generation of Cerebellar Organoids for Ataxia Research grant LISBOA-01-0145-FEDER-029298. Funding was also received from the European Union&#39;s Horizon 2020 Research and Innovation Programme, under the Grant Agreement number 739572&#8212;The Discoveries Centre for Regenerative and Precision Medicine H2020-WIDESPREAD-01-2016-2017.

1. Passaging and maintenance of human iPSCs in monolayer culture
<ol>
	<li>Preparation of plates
	<ol>
		<li>Thaw the basement membrane matrix (see Table of Materials) stock at 4 &#176;C and prepare 60 &#956;L aliquots. Freeze the aliquots at -20 &#176;C.</li>
		<li>To coat the wells of a 6 well plate, thaw one aliquot of the basement membrane matrix on ice. Once thawed add 60 &#181;L to 6 mL of DMEM-F12. Gently resuspend by pipetting up and down.</li>
		<li>Add 1 mL of diluted basement membrane matrix...

<ol>
	<li>Takahashi, 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=Induction+of+Pluripotent+Stem+Cells+from+Adult+Human+Fibroblasts+by+Defined+Factors.">Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts.">Embryonic Stem Cell Lines Derived from Human Blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Lancaster, M. 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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=Human+stem+cells+as+a+model+of+motoneuron+development+and+diseases.">Human stem cells as a model of motoneuron development and diseases.</a> Annals of the New York Academy of Sciences. 1198, 192-200 (2010).</li><li>Mariani, J., Coppola, G., Pelphrey, K. A., Howe, J. R., Vaccarino, F. 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M., Wong, M. M. K., Vowles, J., Cowley, S. A., Becker, E. B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified+Method+for+Generating+Purkinje+Cells+from+Human-Induced+Pluripotent+Stem+Cells.">A Simplified Method for Generating Purkinje Cells from Human-Induced Pluripotent Stem Cells.</a> The Cerebellum. 17 (4), 419-427 (2018).</li><li>Wang, S., 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=Differentiation+of+human+induced+pluripotent+stem+cells+to+mature+functional+Purkinje+neurons.">Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.</a> Science Reports. 5, 9232 (2015).</li><li>Tao, O., 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=Efficient+generation+of+mature+cerebellar+Purkinje+cells+from+mouse+embryonic+stem+cells.">Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells.</a> Journal of Neuroscience Research. 88 (2), 234-247 (2010).</li><li>Croughan, M. S., Giroux, D., Fang, D., Lee, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Single-Use+Bioreactors+for+Scale-Up+of+Anchorage-Dependent+Cell+Manufacturing+for+Cell+Therapies.">Novel Single-Use Bioreactors for Scale-Up of Anchorage-Dependent Cell Manufacturing for Cell Therapies.</a> Stem Cell Manufacturing. , 105-139 (2016).</li><li>Simunovic, M., Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoids,+organoids+and+gastruloids:+new+approaches+to+understanding+embryogenesis.">Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis.</a> Development. 144 (6), 976-985 (2017).</li><li>Lou, Y. R., Leung, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+organoids+for+biomedical+research+and+applications.">Next generation organoids for biomedical research and applications.</a> Biotechnology Advances. 36 (1), 132-149 (2018).</li><li>Silva, T. P., 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=Design+Principles+for+Pluripotent+Stem+Cell-Derived+Organoid+Engineering.">Design Principles for Pluripotent Stem Cell-Derived Organoid Engineering.</a> Stem Cells International. 2019, 1-17 (2019).</li><li>Silva, T. P., 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=Maturation+of+human+pluripotent+stem+cell-derived+cerebellar+neurons+in+the+absence+of+co-culture.">Maturation of human pluripotent stem cell-derived cerebellar neurons in the absence of co-culture.</a> Frontiers of Bioengineering and Biotechnology. , (2020).</li><li>Burridge, P. W., 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=A+universal+system+for+highly+efficient+cardiac+differentiation+of+human+induced+pluripotent+stem+cells+that+eliminates+interline+variability.">A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.</a> PLoS One. 6 (4), 18293 (2011).</li><li>Watanabe, 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=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>Smith, J. 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L., 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=Control+of+Human+Embryonic+Stem+Cell+Colony+and+Aggregate+Size+Heterogeneity+Influences+Differentiation+Trajectories.">Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories.</a> Stem Cells. 26 (9), 2300-2310 (2008).</li><li>Bratt-Leal, A. M., Carpenedo, R. L., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+embryoid+body+microenvironment+to+direct+embryonic+stem+cell+differentiation.">Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation.</a> Biotechnology Progress. 25 (1), 43-51 (2009).</li><li>Miranda, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+up+a+chemically-defined+aggregate-based+suspension+culture+system+for+neural+commitment+of+human+pluripotent+stem+cells.">Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.</a> Biotechnology Journal. 11 (12), 1628-1638 (2016).</li><li>Bardy, 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=Microcarrier+Suspension+Cultures+for+High-Density+Expansion+and+Differentiation+of+Human+Pluripotent+Stem+Cells+to+Neural+Progenitor+Cells.">Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells.</a> Tissue Engineering Part C Methods. 19 (2), 166-180 (2013).</li><li>Rigamonti, 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=Large-Scale+Production+of+Mature+Neurons+from+Human+Pluripotent+Stem+Cells+in+a+Three-Dimensional+Suspension+Culture+System.">Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.</a> Stem Cell Reports. 6 (6), 993-1008 (2016).</li><li>Arora, 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=A+process+engineering+approach+to+increase+organoid+yield.">A process engineering approach to increase organoid yield.</a> Development. 144 (6), 1128-1136 (2017).</li><li>Gimeno, L., Martinez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+chick+Fgf19+and+mouse+Fgf15+orthologs+is+regulated+in+the+developing+brain+by+Fgf8+and+Shh.">Expression of chick Fgf19 and mouse Fgf15 orthologs is regulated in the developing brain by Fgf8 and Shh.</a> Developmental Dynamics. 236 (8), 2285-2297 (2007).</li></ol>

Authors YH and SJ are employees of PBS Biotech. The author BL is CEO and co-founder of PBS Biotech, Inc. These collaborating authors participated in the development of the bioreactors used in the manuscript. This does not alter the authors&#8217; adherence to all the policies of the journal on sharing data and materials. All other authors declare no conflict of interest.

The need for large cell numbers as well as defined culture conditions to generate specific cell types for drug screening and regenerative medicine applications has been driving the development of scalable culture systems. In recent years, several groups have reported the scalable generation of neural progenitors and functional neurons32,33,34, providing significant advances in the development of new models for neurodegenerative ...

The emergence of human pluripotent stem cells (PSCs) represents a excellent tool for regenerative medicine and disease modeling, because these cells can be differentiated into most cell lineages of the human body1,2. Since their discovery, PSC differentiation using diverse approaches has been reported to model different diseases, including neurodegenerative disorders3,4,5,6.
Recently, there have been reports of 3D cultures derived from PSCs resembling human cerebral structures; these are called brain organoids3,7,8. The generation of these structures from both healthy and patient-specific PSCs provides a valuable opportunity to model human development and neurodevelopmental disorders. However, the methods used to generate these well-organized cerebral structures are difficult to apply for their large-scale production. To produce structures that are large enough to recapitulate tissue morphogenesis without necrosis inside the organoids, protocols rely on the initial neural commitment in static conditions, followed by encapsulation in hydrogels and subsequent culture in dynamic systems3. However, such approaches may limit the potential scale-up of organoid production. Even though efforts have been made to direct PSC differentiation to specific regions of the central nervous system, including cortical, striatal, midbrain, and spinal cord neurons9,10,11,12, the generation of specific brain regions in dynamic conditions is still a challenge. In particular, the generation of mature cerebellar neurons in 3D structures has yet to be described. Muguruma et al. pioneered the generation of culture conditions that recapitulate early cerebellar development13 and recently reported a protocol that allows for human embryonic stem cells to generate a polarized structure reminiscent of the first trimester cerebellum7. However, the maturation of cerebellar neurons in the reported studies requires the dissociation of the organoids, sorting of cerebellar progenitors, and coculture with feeder cells in a monolayer culture system7,14,15,16. Therefore, the reproducible generation of the desired cerebellar organoids for disease modeling under defined conditions is still a challenge associated with culture and feeder source variability.
This protocol presents optimal culture conditions for 3D expansion and efficient differentiation of human PSCs into cerebellar neurons using single-use vertical wheel bioreactors (see Table of Materials for specifications), hereafter called bioreactors. Bioreactors are equipped with a large vertical impeller, which in combination with a U-shaped bottom, provide a more homogeneous shear distribution inside the vessel, allowing gentle, uniform mixing and particle suspension with reduced agitation speeds17. With this system, shape and size-controlled cell aggregates can be obtained, which is important for a more homogeneous and efficient differentiation. Moreover, a larger number of iPSC-derived organoids can be generated in a less laborious manner.
The main feature of the organoids, which are 3D multicellular structures usually formed from stem cells, is the self-organization of different cell types that forms specific shapes like those seen in human morphogenesis18,19,20. Therefore, organoid morphology is an important criterion to be evaluated during the differentiation process. Cryosectioning of organoids and further immunostaining of organoid slices with a specific set of antibodies allow for the spatial visualization of molecular markers to analyze cell proliferation, differentiation, cell population identity, and apoptosis. With this protocol, by immunostaining organoid cryosections, an initial efficient neural commitment is observed by the 7th day of differentiation. During differentiation, several cell populations with cerebellar identity are observed. After 35 days in this dynamic system, the cerebellar neuroepithelium organizes along an apicobasal axis, with an apical layer of proliferating progenitors and basally located postmitotic neurons. During the maturation process, from days 35&#8211;90 of differentiation, distinct types of cerebellar neurons can be seen, including Purkinje cells (Calbindin+), granule cells (PAX6+/MAP2+), Golgi cells (Neurogranin+), unipolar brush cells (TBR2+), and deep cerebellar nuclei projection neurons (TBR1+). Also, a nonsignificant amount of cell death is observed in the generated cerebellar organoids after 90 days in culture.
In this system, human iPSC-derived organoids mature into different cerebellar neurons and survive for up to 3 months without the need for dissociation and feeder coculture, providing a source of human cerebellar neurons for disease modeling.

<table>
	<tbody>
		<tr>
			<td>3MM paper</td>
			<td>WHA3030861</td>
			<td>Merck</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>A6964 - 500mL</td>
			<td>Sigma</td>
			<td>cell detachment medium</td>
		</tr>
		<tr>
			<td>Anti-BARHL1 Antibody</td>
			<td>HPA004809</td>
			<td>Atlas Antibodies</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Calbindin D-28k Antibody</td>
			<td>CB28</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 Antibody</td>
			<td>M4403</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-N-Cadherin Antibody</td>
			<td>610921</td>
			<td>BD Transduction</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NESTIN Antibody</td>
			<td>MAB1259-SP</td>
			<td>R&#38;D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-OLIG2 Antibody</td>
			<td>MABN50</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAX6 Antibody</td>
			<td>PRB-278P</td>
			<td>Covance</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SOX2 Antibody</td>
			<td>MAB2018</td>
			<td>R&#38;D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TBR1 Antibody</td>
			<td>AB2261</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TBR2 Antibody</td>
			<td>ab183991</td>
			<td>Abcam</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TUJ1 Antibody</td>
			<td>801213</td>
			<td>Biolegend</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>T1147</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<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>Coverslips 24x60mm</td>
			<td>631-1575</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallization-purified BSA</td>
			<td>5470</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>10236276001</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutyryl cAMP</td>
			<td>SC- 201567B -500mg</td>
			<td>Frilabo</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>Glass Copling Jar</td>
			<td>E94</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax I</td>
			<td>10566-016</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>MB014001</td>
			<td>NZYtech</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;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>Mowiol</td>
			<td>475904</td>
			<td>Millipore</td>
			<td>mounting medium</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 Y-27632</td>
			<td>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>SuperFrost Microscope slides</td>
			<td>12372098</td>
			<td>ThermoFisher</td>
			<td>adhesion microscope slides</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>25608-930</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL 1M</td>
			<td>T3038-1L</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>9002-93-1</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>P1379</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>15575020</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
	</tbody>
</table>

scalable generation of mature cerebellar organoids from human pluripotent stem cells and characterization by immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

The protocol was initiated by promoting cell aggregation using the 0.1 L bioreactors (Figure 1A). Single cell inoculation of the iPSCs was performed, with 250,000 cells/mL seeded in 60 mL of medium with an agitation speed of 27 rpm. This was defined as day 0. After 24 h, the cells efficiently formed spheroid-shaped aggregates (day 1, Figure 1B), and the morphology was well-maintained until day 5, with a gradual increase in size, demonstrating a high degree of ho...

Watch this Scientific Journal Video about Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining at JoVE.com

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

We present for the first time, a new approach for reproducible and scalable generation, of IPS cells derived at Cerebellar Organoids, and a chemical defined conditions, using single use bioreactors. To obtain cells for seeding the bio-reactor, add one milliliter of cell detachment medium, to each well of a six well plate of human iPSC's. Incubate the plate at 37 degrees Celsius for seven minutes, until gentle shaking easily detaches the cells, from the well.Using a P 1000 micropipette, pipet the cell detachment medium, up and down, until the cells dissociate into single cells. Next add two milliliters, of complete cell culture medium to each well, this will inactivate enzymatic digestion. Then use the pipette, to gently transfer the cells to a sterile conical tube.Centrifuge the tube at 210 times gravity for three minutes. Remove the supernatant, and re-suspend the cell pellet in culture medium. Count the cells using a hemocytometer, and trypan blue dye.See the bioreactor vessel with the iPSC's, at a density of 250, 000 cells per milliliter. Insert the bioreactor vessel, into the Universal Base Unit, in an incubator at 37 degrees Celsius, 95%humidity and 5%carbon dioxide. To promote iPSC aggregation, set the agitation rate of the Base Control Unit to 27 RPM.On day one, with day zero 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 one 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 40 X or 100 X.Continue culturing the iPSC's and the bioreactor, until the average diameter of the aggregates, is 100 micrometers, then replace 80%of the medium, with fresh MT01 without rock inhibitor. When the aggregates, reach 200 to 250 micro meters 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.After removing the supernatant from the stored organoids, add one milliliter of 15%sucrose to the pellet, and mix well by swirling gently. After incubating the organoids overnight, at four degrees Celsius, remove the sucrose solution, then add one milliliter of 15%sucrose, 7.5%gelatin, and quickly mix by gently swirling. While the organoids are incubating at 37 degrees Celsius fill a plastic container halfway with the 15%sucrose 7.5%gelatin solution, and allow it to solidify.After the organoids have been incubating for one hour, use a Pasteur pipette, to place a drop of the organoid sucrose gelatin mixture, on top of the solidified sucrose and gelatin. After waiting 15 minutes for the drop to solidify, fill the remainder of the plastic container, with more 15%sucrose, 7.5%gelatin, allow it to solidify completely at room temperature and then incubate it for 20 minutes at four degrees Celsius. Cut the gelatin into a cube, with the organoids in the center, fix the cube to a piece of cardboard, with a drop of OCT compound, then placed 250 milliliters of Isopentane, in a 500 milliliter cup, fill an appropriate container, with liquid nitrogen.Using forceps and thick gloves, carefully place the cup of Isopentane, on the surface of the liquid nitrogen. Isopentane and liquid nitrogens, are hazardous reagents. The use of these two reagents, require personal protective equipment, including thick gloves and an adequate ventilation.When the Isopentane, has cooled to negative 80 degrees Celsius, place the gelatin cube in the Isopentane until it freezes. A well frozen cube, produces a metallic sound when lightly tapped with forceps, indicating that it is ready to be stored. Wash the microscope slides containing the organoid sections, with 50 milliliters 1X PBS for five minutes, then transfer the slides to a Coplin jar, containing fresh 1X PBS.Transfer the slides to a Coplin jar, containing 50 milliliters of freshly prepared glycine and incubate for 10 minutes at room temperature, then transfer the slides to a Coplin jar, containing 50 milliliters of 0.1%Triton, and permeabilized for 10 minutes at room temperature. Wash the slides twice with 1X PBS, for five minutes each time. Prepare the immunostaining dish, with three millimeter paper, soaked in 1X PBS.Dry the slides with a tissue all around the slices and place them on the three millimeter paper. With a Pasteur pipette, cover each slide with 0.5 milliliters of blocking solution. After incubating for 30 minutes at room temperature, remove excess blocking solution and drive the slides with a tissue, all around the slices.Place 50 microliters of diluted primary antibody, on each slide and cover them with cover slips. Lace the slides, in the previously prepared immunostaining dish, and incubate them at four degrees Celsius. After overnight incubation, transfer the slides to a Coplin jar, with 50 milliliters of TBST, letting the cover slips fall off, then wash the slides three times with TBST, for five minutes each time.Place 50 microliters, of diluted secondary antibody on each slide, and cover with the cover slips. Place the slides, in the previously prepared immunostaining dish, incubate for 30 minutes at room temperature, protected from light. Transfer the slides to a Coplin jar again, and wash them three times with 50 milliliters of TBST, for five minutes each time.Using a Pasteur Pipette, at 0.5 milliliters of dappy solution, over the whole surface of each slide and incubate for five minutes at room temperature. After 24 hours in the bioreactor, iPSC's efficiently formed spheroid shaped aggregates. The morphology was well-maintained until day five, with a gradual increase in size.Demonstrating a high degree of homogeneity. A quantitative analysis by microscopy, also revealed normal distribution, of aggregate sizes by day one, and both cell lines attained the optimal aggregate size by day two. After the desired aggregate diameter was achieved, neural commitment was induced, and then generation, of different cerebellar progenitors was promoted.During differentiation organoids, showed a more pronounced epithelialization similar to neural tube-like structures with luminal space. Additionally, the organoid diameter distribution, was homogeneous during the initial cerebellar commitment until day 14. Immunofluorescence analysis supports, that an efficient neural commitment, of the iPSC derived organoids, is already achieved, by day seven of differentiation.Immunostaining also demonstrated, efficient cerebellar commitment and efficient maturation of cerebellar organoids. Furthermore, after 80 days in the bioreactor, live dead staining of organoids showed high cell viability, and no evidence of necrotic areas. This technique represents an important tool, for studying pathological pathways, involved in the degeneration of the cerebellar observed in of and for evaluating the therapeutic effect of new drugs.

scalable-generation-mature-cerebellar-organoids-from-human

Research

JoVE Journal

Bioengineering

10.2K visualizações.  Universidade de Lisboa. Apresentamos pela primeira vez, uma nova abordagem para geração reprodutível e escalável, de células IPS derivadas de Organoides Cerebelares, e condições químicas definidas, utilizando bioreatores de uso único. Para obter células para semear o bio-reator, adicione um mililitro de meio de desprendimento celular, a cada poço de uma placa de seis poços de iPSC humanos. Incubar a placa a 37 graus Celsius por sete minutos, até que o tremor suave se desprende facilmente das células, d...