The effect of cell-cell contact on gene transfection is mainly unknown. Usually, transfection is carriedout in batch cell cultures without control over cellular interactions, and efficiency analysis relies on complex and expensive protocols commonly involving flow cytometry as the final analytical step. Novel platforms and cell patterning are being studied in order to control cellular interactions and improve quantification methods. In this work, we report the use of surface patterning of fibronectin for the generation of two types of mesenchymal stromal cells patterns: single cell patterns without cell-to-cell contact, and small cell-colony patterns. Both scenarios allowed the integration of the full transfection process and the continuous monitoring of thousands of individualized events by fluorescence microscopy. Our results showed that cell-to-cell contact clearly affected the transfection, as single cells presented a maximum transfection peak 6 hours earlier and had a 10 % higher transfection efficiency than cells with cell-to-cell contact.