A novel direct cell printing technique has been developed to control and manipulate the position of cells on solid surfaces. The method utilizes microfabricated polymeric “quill-pen” cantilevers to transfer living cells onto a wide variety of surfaces. In contrast with existing cell deposition methods, such as ink jet or laser ablation methods, the quill-pen approach imparts minimal thermal and shear stress to cells, preserving cell viability and biological functionality. Deposition of both bacterial and mammalian cells into defined patterns has been demonstrated using this method. The size of printed, cell-containing droplets could be controlled by varying the geometry of the quill-pen stylus and by varying printing conditions such as contact time, relative humidity, and surface hydrophobicity. Initial experiments using 10 μm diameter polymer beads demonstrated that the number of beads per droplet could be controlled by varying spot size and particle concentration in the printing solution. Spots could be printed ranging from 20 μm and 100 μm in diameter with approximate volumes ranging from 1-250 pL. We demonstrated deposition of both cells and beads onto a variety of solid surfaces including agarose gel, polystyrene, polyethylene, and glass. Printed cells have also been immobilized on glass and polymer surfaces using biocompatible hydrogel materials (both alginic acid and hyaluronic acid-based matrices) as well as poly-L-lysine. Similar to polymer beads, the number of cells in printed droplets was shown to be dependent upon the size of the droplet, and could be varied by adjusting the concentration of cells present in the printing fluid. As few as one cell per spot could be achieved by adjusting these parameters. The viability and proliferation of printed cells has been evaluated using live optical imaging to observe cell growth and division. Both bacterial cells (Escherichia coli) and mammalian cells were able to divide and proliferate for at least 96 hr post-printing (experiments were discontinued after 96 hr). Live/dead staining was also used to confirm the viability of printed cells. Rat mammary adenocarcinoma MTLn3 cells and mouse embryonic stem cells were also shown to survive the printing process for at least 24 - 96 hr post-printing. These results demonstrate the feasibility of the printing method and its compatibility with a wide range of cell types. It is especially noteworthy that embryonic stem cells could survive the printing process (and proliferate on the printing substrate). This novel printing method has applications for tissue engineering, cell-to-cell signaling studies, and for directly interfacing cells with nanodevices and biosensors.
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