TY - JOUR
T1 - Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies
AU - Buentello, David Choy
AU - Koch, Lena Sophie
AU - Trujillo-De Santiago, Grissel
AU - Alvarez, Mario Moisés
AU - Broersen, Kerensa
N1 - Funding Information:
This work was financially supported by the Netherlands Organ-on-Chip Initiative (NOCI) and By NWO (Dutch Research Council) – Gravity and Alzheimer Nederland grants. DCB thankfully acknowledge the financial support of Consejo Nacional de Ciencia y Tecnología (CONACyT) in the form of a doctoral scholarship. GTdS and MMA gratefully acknowledge the financial support from CONACyT (SNI scholarships 26048 and 256730) and the Biocodex Foundation, México. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Carla Cofiño Fabres for providing V-bottom well plates. We also thank Mariana Garcia-Corral and Carla Annink for their support in cell culture techniques and standardization. We acknowledge the contribution of Joost Le Feber in the analysis of the electrophysiological data.
Publisher Copyright:
Copyright: © 2022 Choy Buentello et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2022/5/10
Y1 - 2022/5/10
N2 - 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 ×103 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 11×103 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 7×103 and 11×103 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.
AB - 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 ×103 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 11×103 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 7×103 and 11×103 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.
UR - https://www.scopus.com/pages/publications/85130001569
U2 - 10.1371/journal.pone.0262062
DO - 10.1371/journal.pone.0262062
M3 - Article
C2 - 35536781
AN - SCOPUS:85130001569
SN - 1932-6203
VL - 17
JO - PLoS ONE
JF - PLoS ONE
IS - 5 May
M1 - e0262062
ER -