Step-minimized EDTA-based detachment protocol for human pluripotent stem cells cultured on laminin-511 E8 fragment

The recombinant laminin-511 E8 fragment (LM511E8) is a highly effective, xeno-free matrix for human pluripotent stem cell (hPSC) culture. However, the firm adhesion of hPSCs to LM511E8 conventionally requires mechanical scraping after enzymatic treatment, thereby compromising cell viability, reproducibility, and culture scalability.

We developed a step-minimized, ethylenediaminetetraacetic acid (EDTA)-based, scraper-free detachment protocol for hPSCs cultured on LM511E8. The protocol eliminates both mechanical scraping and centrifugation, and was evaluated for detachment efficiency, long-term maintenance of hPSC quality, applicability to mid-scale culture systems, and the effects of trypsin-like proteases on EDTA-mediated cell detachment.

The protocol achieved consistently high detachment efficiency with minimal handling while preserving hPSC pluripotency and genomic integrity over long-term culture. Notably, the addition of trypsin-like proteases to the EDTA solution reduced detachment efficiency, which was restored by protease inhibition. Furthermore, the method was readily adaptable to mid-scale culture systems (e.g., multilayer flasks) with minor modifications.

Our protocol provides an efficient, reproducible, and centrifugation-free detachment method that resolves the technical limitations of LM511E8-based cultures. This scalable approach may facilitate high-throughput hPSC expansion in both basic research and regenerative medicine applications.