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Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies
The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9089918/ https://www.ncbi.nlm.nih.gov/pubmed/35536781 http://dx.doi.org/10.1371/journal.pone.0262062 |
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author | Choy Buentello, David Koch, Lena Sophie Trujillo-de Santiago, Grissel Alvarez, Mario Moisés Broersen, Kerensa |
author_facet | Choy Buentello, David Koch, Lena Sophie Trujillo-de Santiago, Grissel Alvarez, Mario Moisés Broersen, Kerensa |
author_sort | Choy Buentello, David |
collection | PubMed |
description | The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×10(3) cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×10(3) cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×10(3) and 11×10(3) and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages. We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research. |
format | Online Article Text |
id | pubmed-9089918 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-90899182022-05-11 Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies Choy Buentello, David Koch, Lena Sophie Trujillo-de Santiago, Grissel Alvarez, Mario Moisés Broersen, Kerensa PLoS One Research Article The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×10(3) cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×10(3) cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×10(3) and 11×10(3) and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages. We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research. Public Library of Science 2022-05-10 /pmc/articles/PMC9089918/ /pubmed/35536781 http://dx.doi.org/10.1371/journal.pone.0262062 Text en © 2022 Choy Buentello et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Choy Buentello, David Koch, Lena Sophie Trujillo-de Santiago, Grissel Alvarez, Mario Moisés Broersen, Kerensa Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies |
title | Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies |
title_full | Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies |
title_fullStr | Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies |
title_full_unstemmed | Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies |
title_short | Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies |
title_sort | use of standard u-bottom and v-bottom well plates to generate neuroepithelial embryoid bodies |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9089918/ https://www.ncbi.nlm.nih.gov/pubmed/35536781 http://dx.doi.org/10.1371/journal.pone.0262062 |
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