Endpoint gastruloid-on-microraft analysis (EGOMRA)

Although adherent two-dimensional human gastruloids are highly
reproducible and provide insights into early embryogenesis, no available
automated technology can screen and sort large numbers of these
near-millimeter-sized complex structures for large-scale assays.
Consequently, we developed a microraft array-based technology to perform
image-based assays of large numbers of fixed or living gastruloids and
sort individual gastruloids for downstream assays, such as gene expression
analysis. We created datasets with metrics describing the microraft arrays
and the templates used to make the arrays, micropatterning of
extracellular matrix (ECM) to form gastruloids, gastruloid formation, and
gastruloids formed on both multi-well plates and microraft arrays. The
microraft array platform was used to assay individual euploid and
aneuploid (possessing abnormal numbers of chromosomes) gastruloids with
clear phenotypic and gene expression differences. Both noggin (NOG) and
keratin 7 (KRT7), two genes involved in spatial patterning within
gastruloids, were upregulated in aneuploid relative to that in the euploid
gastruloids. Moreover, relative NOG and KRT7 expressions were negatively
correlated with DNA/area. The microraft arrays will empower novel screens
of single gastruloids for a better understanding of key mechanisms
underlying phenotypic differences between gastruloids.