Reproducibility assessment of the hiPSC-derived Brain Spheroid model across multiple laboratories and cell lines

However, their broader adoption critically depends on reproducibility and standardization. Here, we present a multi-site evaluation of the BrainSphere model, a three dimensional central nervous system microtissue derived from hiPSCs, conducted across three international laboratories using five hiPSC lines and two protocols to generate neural progenitor cells, which serve as the starting material for BrainSphere formation.

Despite differences in cell line origin, passage number, and laboratory equipment, BrainSpheres consistently developed into mixed neural populations, including neurons, astrocytes, and oligodendrocytes. While single cell RNA sequencing revealed differences in cell type proportions and transcriptional states between lines, immunohistochemistry and proteomics consistently confirmed the presence of these key populations across all sites.