Commercial dishes with gelatin-free microstructured inserts for elongated stem cell self-renewal and pluripotency

Here, we report the scalable fabrication of 2i-functionalized micro-pyramid-array (mu PyA/+2i) inserts for use in commercial multi-well plates, as the alternative culti-vation platform for maintaining long-term self-renewal and pluripotency of mul-tiple mESCs and mouse induced pluripotent stem cells. Relevant evidence including cell morphology characterization increased alkaline phosphatase activ-ity, high expression of mESC self-renewal markers, decreased levels of differen-tiation-associated markers, and high proportion of self-renewal marker cells are provided. Further studies demonstrated that mu PyA/+2i could cause a higher cell density in mESC colony, and induce gene expression changes. Subsequent studies showed that mu PyA/+2i can influence the cytoskeleton and promote cell adhesion through Cldn-7 upregulation. In summary, these mu PyA/+2i inserts offer flexible and gelatin-free micro-envriomnets to maintain long-term self-renewal and pluripotency of mESCs. Enabled by the microstructured inserst, the facile stem cell manipulation and transfer among culture dishes will broaden stem cells both in routine and translational applications.

Automated summary

Here, we report the scalable fabrication of 2i-functionalized micro-pyramid-array (mu PyA/+2i) inserts for maintaining long-term self-renewal and pluripotency of multiple mESCs. These inserts provided flexible and gelatin-free microenvironments and showed increased alkaline phosphatase activity, high expression of mESC self-renewal markers, decreased levels of differentiation-associated markers, and high proportion of self-renewal marker cells. The microstructured inserts also influenced the cytoskeleton and cell adhesion through Cldn-7 upregulation, enabling easier stem cell manipulation and transfer among culture dishes.