Thin stencil membrane-assisted high throughput single-cell to cluster of cells micropatterning and large-size biomolecular transfection in primary and stem cells

Abstract

Precise control of cellular organization and intracellular delivery is essential for bioengineering and therapeutic applications. We present an integrated platform that combines cell micropatterning using PDMS stencil membranes with a micropatterned rGO device for photoporation. 30 μm-thick PDMS stencil membranes (with microhole diameters ranging from 25 μm to 100 μm and a 2 cm × 2 cm area) enable scalable, reproducible creation of single- and multicellular clusters with high efficiency. We achieved 100% patterning efficiency for microhole sizes of 100 μm, 80 μm, and 60 μm. With 25 μm holes, we achieved single-cell patterning with ∼90% efficiency for SiHa cells. The array of micropatterned rGO structures (10 μm circular rGO with an interspacing of 30 μm) acts as a photothermal platform. When activated by nanosecond-pulse laser scanning, it temporarily permeabilizes cell membranes, allowing rapid biomolecular delivery into cells. We transfected cellular microconstructs of different cell types (L929, SiHa, LN229, HDFs, and hMSCs) with small to very large biomolecules, such as PI dye (668 Da), siRNA (20–24 bp), EGFP (229 kDa), and enzymes (464 kDa). The best EGFP transfection was achieved in human dermal fibroblasts (HDFs) and human mesenchymal stem cells (hMSCs) at 89% and 93%, with cell viabilities of 92% and 96%. Enzyme transfection of HDFs and hMSCs achieved 88% efficiency, with cell viabilities of 90% and 94%, respectively. Our platform provides high-throughput, geometry-dependent cell patterning with precise delivery, supporting advanced cell biology research and the creation of spatially engineered in vitro models for biomedical research.