Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Abstract

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication. [1][2][3][4][5][6][7][8][9][10] Recently, thermal inkjet printing has also been used for gene transfection. 8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm. 8,9,11 The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells. 8,12 Cell viability after printing has been shown to be similar to standard cell plating methods 1,8 . In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11 A standard HP DeskJet 500 printer was modified to allow for cell printing. 3,5,8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications. 13-15

Session I – Biofabrication (V01–V05)

“…B. et al (2012, [3]). 150 µl of a single cell suspension (Hep G2, 30*10 6 /ml) was transferred into the printing cartrigde.…”

The Integration of Nanotechnology and Biology for Cell Engineering: Promises and Challenges

“…In a novel strategy, a commercial inkjet printer was employed for bioprinting fibroblasts on a 3D substrate. The shear stress applied during the droplet formation led to the development of transient pores about 10 nm in diameter that were used to introduce fluorescent actin into the cells that aided in the visualization of the cytoskeleton dynamics [117]. The results from this study have opened up new avenues for the nanopatterning of extracellular matrix proteins onto specific locations on a substrate, which can provide unique cellular microenvironments for tissue engineering applications.…”

Empowering Mesenchymal Stem Cells for Ocular Degenerative Disorders

“…Furthermore, co-printing using a thermal-based approach may serve as an efficient transfection tool to deliver therapeutic agents which may influence cell survivability, proliferative, and regenerative capabilities [66,67,68]. Cui et al [66] previously observed that cell printing causes a transient pore opening on the cell membrane of printed cells, which was found to facilitate transfection of plasmid encoding for green fluorescent protein (GFP) without compromising cell viability [67,68]. This strategy can be employed on MSCs to achieve a successful delivery system of functional genes or nanocarriers that minimizes issues related to cell incompetency and toxicity [69,70].…”