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.

Keywords: Injection-dose response; Lance array nanoinjection; Serial injection; Speed of injection.

Fig. 1

SEM image of two rows of lances contained on the lance array silicon chip. Lances measure 10 μm in length, 1–2.5 μm in diameter, and spaced 10 μm from center to center

Fig. 2

Lance array nanoinjection stepwise process. 1 Staging the lance array in the solution containing the desired molecular load. 2 Electrical attraction of the molecular load onto the lances. 3 Physically penetrating the cell membrane of target cells and electrical repulsion of the molecular load into the cytoplasmic space. 4 Removal of the lance array, leaving the molecular load in the intracellular space of target cells

Fig. 3

Cross-sectional schematic of injection device. Components include (top–bottom): stepper motor, threaded rod, coiled and orthoplanar springs, electrical connections, silicon lance array, glass slide for cell culture, and cell culture platform

Fig. 4

Experimental set-up showing the electrical control box receiving three separate input signals coming from three power supplies (not shown) and outputting appropriately timed output signals to the injection device mounted above the prepared six-well plate. Cell culture platforms with the prepared cell cultures are seen as white and red circular components resting in the wells of the six-well plate

Fig. 5

Electrical schematic for the current control box. An Arduino was used to control two relays and a stepper motor driver for the injection process. Five LEDs are used as indicators for power, output, and which input being passed through the box

Fig. 6

Injection speed box plot. The two left-most box plots were the result of the slower stepper motor (Rohs) whereas the three right-most box plots were the result of the faster stepper motor (AA). Statistically significant relationships are noted with an asterisk

Fig. 7

Mean percentage of living/propidium iodide positive HeLa cells for all sample types. Because of the number of statistical relationships that were derived, statistically significant relationships are not noted on the box plot figure. For statistical significant relationships, reference Table 3

Fig. 8

Mean percentage of living/propidium iodide positive fibroblast cells for all sample types. Because of the number of statistical relationships that were derived, statistically significant relationships are not noted on the box plot figure. For statistical significant relationships, reference Table 3