Ultrathin SU-8 membrane for highly efficient tunable cell patterning and massively parallel large biomolecular delivery

Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 μm. It features an array of micro-holes ranging from 40 μm to 80 μm, spaced apart by 50 μm to 150 μm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 μm outer diameter, 3 μm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and β-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications.