A Bidirectional Nanotube Stamping Platform for Organelle-Scale Intracellular Delivery into Suspension Cells

Abstract

In regenerative medicine, cell therapy often relies on suspension cells, creating an urgent need for efficient delivery of multiple cargoes into these cells. Our previous nanotube (NT) stamping platform enabled efficient delivery into adherent cells, but delivery into suspension cells─a major therapeutic target─has not yet been realized. Here, we present a versatile NT stamping platform enabling high-efficiency, size-independent delivery into suspension cells without compromising viability. Our platform consists of a vertically aligned gold NT array integrated onto a glass stamp, allowing controlled interfaces with cells under microscopic observation. By optimizing NT geometry and insertion depth, we achieved efficient intracellular delivery of fluorescent dyes, proteins (GFP and LOx), and mitochondria into suspension cells immobilized by a temporary Cell-Tak adhesion strategy, with delivery efficiencies exceeding 90% and cell viability above 85%, respectively. Unlike electroporation, our approach maintains cellular functionality and enables precise, reagent-free delivery. Notably, the platform’s ability to reversibly extract and inject intracellular fluid enabled the direct transfer of stem cell–derived mitochondria into suspension cells, enhancing ATP production per cell by 2-fold for at least 1 week. These results establish NT stamping as a complementary intracellular delivery platform for applications requiring precise delivery of cargoes spanning from small molecules to intact organelles while maintaining high cell viability.