High‐Viability Circulating Tumor Cells Sorting From Whole Blood at Single Cell Level Using Laser‐Induced Forward Transfer‐Assisted Microfiltration

The efficient isolation and molecular analysis of circulating tumor cells (CTCs) from whole blood at single-cell level are crucial for understanding tumor metastasis and developing personalized treatments. The viability of isolated cells is the key prerequisite for the downstream molecular analysis, especially for RNA sequencing. This study develops a laser-induced forward transfer -assisted microfiltration system (LIFT-AMFS) for high-viability CTC enrichment and retrieval from whole blood. The LIFT-compatible double-stepped microfilter (DSMF), central to this system, comprises two micropore layers: the lower layer's smaller micropores facilitate size-based cell separation, and the upper layer's larger micropores enable liquid encapsulating captured cells. By optimizing the design of the DSMFs, the system has a capture efficiency of 88% at the processing throughput of up to 15.0 mL min-1 during the microfilter-based size screening stage, with a single-cell yield of over 95% during the retrieval stage. The retrieved single cells, with high viability, are qualified for ex vivo culture and direct RNA sequencing. The cDNA yield from isolated CTCs surpassed 4.5 ng, sufficient for library construction. All single-cell sequencing data exhibited Q30 scores above 95.92%. The LIFT-AMFS shows promise in cellular and biomedical research.