New technology for cellular piercing: rotationally oscillating μ-injector, description and validation tests

Abstract

ICSI (intracytoplasmic sperm injection) procedure is one of the most commonly used cellular-injection processes. In ICSI a drawn glass pipette is pushed against the biological cell and a series of force impulses are exerted on it axially to achieve the piercing through the zona and the membrane in sequence for the ensuing injection. In most advanced applications a piezo actuator creates this impulsive forcing. This procedure presently requires a very small mercury column inside the glass pipette which is found to be helpful especially for minimizing the transverse oscillations. Despite the toxic mercury, the procedure is commonly utilized in many laboratories. Earlier investigations point out that considerable lateral tip oscillations of the injection pipette remain as the piezo-electric pulses are introduced. Such oscillations damage the cell membrane and impart adverse effects on the success rate of the injection. In this study, we introduce a novel microinjection procedure, which will remedy the shortfalls of the present technology. The highlight of this procedure is the introduction of rotational oscillations to the pipette during the drilling. These oscillations of small amplitudes (few degrees) and high enough frequencies (100 Hz and higher) are shown to create very effective piercing. The so-called Ros-Drill is a mercury-free and minimally invasive device of which the prototypes are designed and built including the relevant peripheral control hardware and software. Preliminary experimental results are presented on mouse oocytes and they are very encouraging. In the early trials on mouse oocytes, several blastocyst stage developments are reported using new drilling device. We also explain in this text the implementation protocols developed for the new technology.

Micro-Nano Technologies for Cell Manipulation and Subcellular Monitoring

“…This is possibly the most sensible step looking forward in the process to understand edge roughness and its consequences on cellular manipulation. To this effect, a number of efforts to introduce current microtechnologies into the context of pipette processing (Kometani et al, -2008Malboubi et al, 2009;Campo et al, 2010a) and piercing techniques (Ergenc & Olgac, 2007) have appeared in the literature. In particular, the use of focus ion beams and electron microscopy may have opened a new avenue to the generation of improved or even, altogether new, tools in the biomedical sciences.…”

“…However, the additional cost of piezo-drills and, more importantly, toxicity issues arising from the use of mercury, have limited the use of piezo-assisted microinjection to just a few species (Yoshida & Perry, 2007;Ergenc et al, 2008). Following this line of research and given the increasing need to improve current cell piercing (Ergenc & Olgac, 2007) and pipette quality (Yaul et al 2008;Ostadi et al 2009), Campo and coworkers have sharpened conventional glass capillaries by FIB and tested them in mouse embryos. For the first time, the effect of sub-nanometer pipette polishing on zona pellucida and plasma membrane piercing is investigated.…”

The Development of Piezo-Driven Tools for Cellular Piercing

“…To realize ICSI, many techniques have been developed such as conventional ICSI [2,9,10], piezo-assisted ICSI [4,11,12], and the Ros-drill technique [13]. Conventional ICSI is commonly implemented in humans and domestic animals where the rupture of the oolemma is achieved by largely deforming the oocyte.…”

“…In order to penetrate the zona pellucida without significantly deforming the oocyte, piezo-driven ICSI and the Ros-drill technique were developed. In the Ros-drill technique, the tip of the injection pipette is rotationally oscillated [13]. This technique produces a cleavage rate of approximately 70% in mouse oocytes.…”

Focus ion beam micromachined glass pipettes for cell microinjection

“…In piezo-driven drilling, a small amount of mercury is typically loaded in the micropipette to minimize lateral vibrations, preventing cell damage (Ediz and Olgac 2005). However, the additional cost of piezo-drills and, more importantly, toxicity issues arising from the use of mercury have limited the use of piezo-assisted microinjection to just a few species (Yoshida and Perry 2007;Ergenc and Olgac 2007).…”