Permeabilization of Adhered Cells Using an Inert Gas Jet

Summary

This protocol describes a method for the temporary permeabilization of adherent cells using an inert gas jet. This technique facilitates the transfer of genetic material and biomolecules into adherent mammalian cells by the utilization of mechanical forces to disrupt the plasma membrane.

Abstract

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes a mechanical method to temporally permeabilize adherent cells using an inert gas jet that can facilitate the transfer of normally non-permeable macromolecules into cells. We believe this technique works by imparting shear forces on the plasma membrane of adherent cells, resulting in the temporary formation of micropores. Once these pores are created, the cells are then permeable to genetic material and other biomolecules. The mechanical forces involved do run the risk of permanently damaging or detaching cells from their substrate. There is, therefore, a narrow range of inert gas dynamics where the technique is effective. An inert gas jet has proven efficient at permeabilizing various adherent cell lines including HeLa, HEK293 and human abdominal aortic endothelial cells. This protocol is appropriate for the permeabilization of adherent cells both in vitro and, as we have demonstrated, in vivo, showing it may be used for research and potentially in future clinical applications. It also has the advantage of permeabilizing cells in a spatially restrictive manner, which could prove to be a valuable research tool.

Introduction

With the evolution of biomedicine and the understanding of cell mechanics, the delivery of biomolecules into cells has become vital to many research fields and medical therapies. Different techniques have been developed to introduce foreign molecules through the plasma membrane and into the cell cytosol. These can be generally classified as either: viral, chemical or mechanical techniques. Viral techniques can transfect genetic material such as RNA and DNA using viral vectors¹. Viral techniques tend to have high efficiencies in certain cell lines, however they do have the potential for immune and inflammatory reactions². Common chemical transfect....

Protocol

1. Development of LabView Program

1. A mass flow controller (MKS M100B Mass-Flo Controller) is attached to the helium gas line to ensure a precise gas flow rate. This unit is controlled by an input voltage, ranging between 0 and 5 V. A control loop in the LabView program determines the required voltage at any given time. Interfacing between the computer and the mass flow controller is performed by a data acquisition device (NI USB-6009), and a 12 V power supply provides power to the mass flow controller.
2. Two positioning linear slides are used to control the movement of the 6-well plate, one for each direction. Each assembly consists of a MA15-series....

Results

Permeabilization of HeLa cells with 10 kDa green fluorescent dextran using a helium gas jet with a 0.86 mm inner diameter capillary is shown in Figure 1. Cells were counterstained immediately after permeabilization with 2 μl/ml EthD-1 solution (LIVE/DEAD Viability/Cytotoxicity Kit) to visualize cell death. Immediately following counterstaining, cover slips were mounted and imaged. This figure shows results of running the helium gas jet at three different outlet pressures compared to a control sample. Not.......

Discussion

Inert gas jet permeabilization is a useful technique for adherent cell transfection. It provides the ability to transfer biomolecules into cells with the use of mechanical forces, which eliminates the need for potentially harmful chemicals or viral vectors. The technique could potentially give researchers and clinicians an efficient and relatively simple way to precisely transfect cells. The selectivity of the permeabilization is also unique allowing researchers to only treat certain cells in a single colony which could .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vitality hrGFPII-1	Agilent Technologies Canada	240143-57	Green fluorescent protein for transfection experiments on HeLa cells
Dextran, AlexaFluor 488; 10,000 MW, Anionic, Fixable	Life Technologies Inc.	D22910	dextran for permeabilization experiments
LIVE/DEAD Viability/Cytotoxicity Kit	Life Technologies Inc.	L3224	for counterstaining of mammalian cells
Prolong Gold Antifade Reagent	Invitrogen	P36930	for mounting slides when imaging
Ultra High Purity Helium	Praxair Canada Inc.	HE 5.0UH-T
Hyclone DMEM/ High Glucose Media - 500 ml	Thermo Scientific	SH30022.01	for HeLa cells
Fetal Bovine Serum	Invitrogen	26140079	For DMEM media
Penicillin-Streptomycin, liquid	Invitrogen	15140-122	For DMEM media
Phosphate-Buffered Saline (PBS) 1x			Produced in lab
			Table 1. List of required reagents
6-Well Clear TC-Treated Microplates	Corning	3506
#1 22 x 22 Coverslips	Fisher	12-542B
Glass Capillaries	World Precision Instruments		WPI Sizing Information
ID (mm): 1, 0.86, 0.68, 0.5 Order #: 1B200, 1B150, 1B120, 1B100
Mass-Flo Controller	MKS	M100B01314CS1BV	Mass flow controller, Series M100B, requires an external 12 V power supply
USB-6009 DAQ Device	National Instruments	779026-01
UniSlide Assembly with stepping motor and controller	Velmex		Two series MA15 UniSlide Assemblies, fitted with Vexta 1.8° stepping motors provided by Velmex, and controlled by a VXM Stepping Motor Controller
			Table 2. List of required equipment