Single‐Cell Nucleus Extraction with Cellular Indexing

Abstract Bulk organelle‑fractionation masks cell‑to‑cell heterogeneity, and existing microfluidic methods cannot reliably reconnect each isolated organelle to its parent cell, an essential capability for multiomics readouts. VacTrap, a high‐throughput microfluidic device that isolates and spatially indexes single nuclei from mammalian cells is developed. The VacTrap device consisted of three aligned layers: 1) a Bis‐gel microwells layer with a “trapdoor” (BAC‐gel) base, fabricated atop a through‐hole glass slide; 2) a polydimethylsiloxane (PDMS) microwell layer to receive transferred nuclei; and 3) a vacuum manifold. VacTrap operation begins with cell cytoplasmic lysis using differential detergent fractionation (DDF) to release intact nuclei into the Bis‐gel microwells, while cytoplasmic proteins are electrophoresed into the Bis‐gel layer. Subsequent addition of dithiothreitol (DTT) and vacuum dissolves the trapdoors within 3–5 min, synchronously transferring nuclei into the PDMS microwells, achieving 98% efficiency across 80% of trapdoors. To verify fractionation of the cytoplasmic proteins from each cell nucleus, select protein targets are successfully detected by in situ immunoprobing in the archival Bis‐gel layer. To verify the fractionation and collection of individual intact nuclei, the morphology analysis confirms preservation of the nuclear features. By introducing spatial indexing of nuclei back to the originating cell, VacTrap provides a robust, automated cell‐preparation platform for single‐cell multiomics applications.