Optimization of embryo vitrification media using stem cell derived human blastoids

Stem cell-derived human blastoids have recently emerged as a new tool in modeling human development. Here, we aim to identify optimal conditions for vitrifying and warming blastoids and assess their potential as a research tool to advance human ART. Human blastoids were created based on an established protocol (Yu et al., bioRxiv, 2022). Good quality blastoids with at least 150 μm diameter (similar in size to a human blastocyst graded stages 3-6), a blastocoel cavity occupying greater than half of the embryo, and approx. 10-20% of the blastoid displaying well-organized ICM-like cells within the blastocoel were selected. Blastoids were vitrified on Cryotop devices and warmed according to the Kitazato protocol using either M199-based vitrification and warming media or N2B27-based vitrification and warming media, which are all prepared in-house. Warmed blastoids were recovered in a culture medium and imaged on a stereoscope to assess survival and re-expansion up to 3 h post-warming. Warmed blastoids were fixed in 4% PFA, stained with DAPI, POU5F1, GATA3, and cleaved Caspase-3, and imaged by confocal microscopy to examine cell lineage distribution and apoptosis. Next, warmed blastoids were subjected to three days of extended culture in idbi μ-Slide 8 Well dishes coated with fibronectin. At the end of extended culture, blastoids outgrowth were stained with DAPI, POU5F1, GATA3, and CGB to assess epiblast and trophoblast development of the blastoids vitrified and warmed in different media. Nearly all blastoids (73/74) survived vitrification and warming, and 91.9% re-expanded within 3 h post-warming. The percent of cell apoptotic cells / total cells was similar between the two medium groups. A total of 16 blastoids were randomly selected to be plated for extended culture. All blastoids attached to the fibronectin plates and the outgrowth area of the trophoblast were similar between the two media groups (M199, 0.15 ± 0.04 mm2, and N2B27, 0.16 ± 0.03 mm2). The total cell number of the blastoid outgrowth identified by DAPI staining was also similar between the two groups (M199, 118.3 ± 28.2, and N2B27, 157.9 ± 21.9). However, the epiblast cell numbers of the blastoid outgrowth in the N2B27 group (96.8 ± 20.2) tend to be more abundant than the ones in the M199 group (42.8 ± 10.3) (P=0.052). On the other hand, CGB-positive trophoblast cells, suggesting the formation of syncytiotrophoblast, were more abundant in the M199 group (58.6% ± 9.3%) than the N2B27 group (33.3% ± 8.8%) (P=0.02). Our results demonstrate that stem cell-derived blastoids can successfully survive vitrification and warming processes and undergo further development during extended culture. The N2B27-based vitrification and warming medium may favor the preservation of epiblast cells during the vitrification process, offering the potential opportunity to optimize the current widely used M199-based vitrification and warming medium.