The effect of injection speed and serial injection on propidium iodide entry into cultured HeLa and primary neonatal fibroblast cells using lance array nanoinjection

Abstract

Background: Although site-directed genetic engineering has greatly improved in recent years, particularly with the implementation of CRISPR-Cas9, the ability to deliver these molecular constructs to a wide variety of cell types without adverse reaction is still a challenge. One non-viral transfection method designed to address this challenge is a MEMS based biotechnology described previously as lance array nanoinjection (LAN). LAN delivery of molecular loads is based upon the combinational use of electrical manipulation of loads of interest and physical penetration of target cell membranes. This work explores an original procedural element to nanoinjection by investigating the effects of the speed of injection and also the ability to serially inject the same sample. Results: Initial LAN experimentation demonstrated that injecting at speeds of 0.08 mm/s resulted in 99.3 % of cultured HeLa 229 cells remaining adherent to the glass slide substrate used to stage the injection process. These results were then utilized to examine whether or not target cells could be injected multiple times (1, 2, and 3 times) since the injection process was not pulling the cells off of the glass slide. Using two different current control settings (1.5 and 3.0 mA) and two different cell types (HeLa 229 cells and primary neonatal fibroblasts [BJ(ATCC® CRL-2522™)], treatment samples were injected with propidium iodide (PI), a cell membrane impermeable nucleic acid dye, to assess the degree of molecular load delivery. Results from the serial injection work indicate that HeLa cells treated with 3.0 mA and injected twice (×2) had the greatest mean PI uptake of 60.47 % and that neonatal fibroblasts treated with the same protocol reached mean PI uptake rates of 20.97 %. Conclusions: Both experimental findings are particularly useful because it shows that greater molecular modification rates can be achieved by multiple, serial injections via a slower injection process.