Fabrication and characterization of glass micropipettes: Exploration of use as single-cell manipulation and bioprinting tips

Abstract

Micropipettes are primarily used for cell transfection procedures (microinjection). However, their use has been limited to cells larger than 25 µm in diameter. Alternatively, nanopipettes have been shown to enable the transfection and manipulation of cells between 7 and 25 µm in diameter. In this work, the fabrication of micro- and nanopipettes through the pulling process is described. Micropipettes ranging in tip diameters from 40 to 170 µm, and nanopipettes as thin as 140 nm in tip diameters were fabricated with high repeatability. The resulting diameter of the tip can be controlled by changing operational parameters during the stretching process such as heating temperature, pulling force, pulling speed, and the capillary glass composition. Tip sizes were characterized by Scanning Electron Microscopy (SEM). In addition, in proof-of-concept experiments, we explored two applications for the fabricated micropipettes and nanopipettes. First, alginate-based hydrogel fibers were extruded through micropipettes as thin as 50 micrometers in diameter. These fibers were used as tissue engineering scaffolds to culture muscle precursor cells. In an additional set of experiments, we use micropipettes as bioink extruders. Alginate-fibers, loaded with fluorescent bacteria (i.e., two distinct Escherichia coli strains engineered to produce red and green fluorescent proteins), were co-extruded through a printhead consisting in a Kenics static mixer coupled to a micropipettes tip to fabricate microstructured bacterial constructs composed of intercalated layers of green and red bacteria. Finally, we illustrate the use of nanopipettes to micro-manipulate adhered muscle culture cells during culture.