Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies

Abstract

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.

Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells

“…Our results highlight the importance of choosing and validating an aggregation method that better suits the desired applications. For instance, different results in terms of EB survival and differentiation performance have been reported for EB formed even for V and U bottom plates [ 55 ].…”

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research

“…However, varying diameters have been shown to influence differentiation rates and success 29 . For reproducibility purposes, diameter variations below 50 µm are highly recommended 20 . Additionally, the number of neural induction days can be extended from 6 to 10 days to allow for the EBs to grow in diameter, as the use of SMAD inhibitors has been shown to efficiently produce and maintain neuroepithelia formation after only 5 days of exposure 30 .…”

“…6. Aspirate the supernatant with a pipette, resuspend the cell pellet in 1 mL of Essential 6 (E6, see 20 ). Seed 9,000-11,000 cells per well.…”

The Analysis of Embryoid Body Formation and Its Role in Retinal Organoid Development

“…A comprehensive summary of this analysis is provided in Table 1. We found three reviews that systematically provided information on the effect of physical features of the culture environment [9,18,19]. The impact of exogenous signals was investigated in three other independent studies [10,12,20].…”