Weekend-free and robotics-compatible protocols for high-throughput human-induced pluripotent stem cell maintenance, amplification, and differentiation.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) are essential tools for disease modeling, drug testing, and regenerative medicine. These applications require differentiation of multiple cell lines in parallel, synchronized production of large numbers of hiPSCs and differentiated cells, and assessment of differentiation efficiency before proceeding to in-depth characterization through molecular and cellular assays. However, hiPSC culture protocols are often laborious manual processes, which affects reproducibility and makes high-throughput applications challenging. METHODS: We describe high-throughput hiPSC maintenance and amplification protocols that are weekend-free, scalable, and can be performed manually or automated. These optimized protocols enable scale-up of hiPSC production and differentiation to multiple cell types, including endothelial cells, microglia, and retinal pigment epithelial cells. RESULTS: We characterized the differentiated cells using molecular and cellular assays and demonstrated that cells generated with this approach represent accurate cell identities. CONCLUSION: The approaches described here enable high-throughput hiPSC applications and improve reproducibility and scalability.If you want, I can also tighten this further to better fit a strict 350-word journal limit or adapt the heading style to a specific journal.