Name: permeabilization of adhered cells using an inert gas jet
Uploaded: 2013-09-04T14:00:00
Description: Watch this Scientific Journal Video about Permeabilization of Adhered Cells Using an Inert Gas Jet at JoVE.com

The authors would like to thank the following funding sources: Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council (NSERC).

1. Development of LabView Program
<ol>
	<li>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.</li>
	<...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+DNA+Transferred+into+Mammalian-Cells.">Expression of DNA Transferred into Mammalian-Cells.</a> J. Bioscience. 6, 543-567 (1984).</li><li>Spandidos, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-mediated+gene+transfer+(electroporation)+into+mouse+Friend+and+human+K562+erythroleukemic+cells.">Electric field-mediated gene transfer (electroporation) into mouse Friend and human K562 erythroleukemic cells.</a> Gene analysis techniques. 4, 50-56 (1987).</li><li>Chu, G., Hayakawa, H., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+for+the+efficient+transfection+of+mammalian+cells+with+DNA.">Electroporation for the efficient transfection of mammalian cells with DNA.</a> Nucleic Acids Research. 15, 1311-1326 (1987).</li><li>Fechheimer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+mammalian+cells+with+plasmid+DNA+by+scrape+loading+and+sonication+loading.">Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 8463-8467 (1987).</li><li>Koch, S., Pohl, P., Cobet, U., Rainov, N. 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S., Burkholder, J., Roberts, B., Martinell, B., McCabe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+gene+transfer+to+mammalian+somatic+cells+by+particle+bombardment.">In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.</a> Proceedings of the National Academy of Sciences of the United States of America. 87, 9568-9572 (1990).</li><li>Hallow, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear-induced+intracellular+loading+of+cells+with+molecules+by+controlled+microfluidics.">Shear-induced intracellular loading of cells with molecules by controlled microfluidics.</a> Biotechnology and Bioengineering. 99, 846-854 (2008).</li><li>Chouinard-Pelletier, G. 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Plasma Sci. 37, 927-933 (2009).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+shear+devices+for+quantitative+analysis+of+cell+adhesion.">Microfluidic shear devices for quantitative analysis of cell adhesion.</a> Analytical Chemistry. 76, 5257-5264 (2004).</li><li>Sankin, G. N., Yuan, F., Zhong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsating+tandem+microbubble+for+localized+and+directional+single-cell+membrane+poration.">Pulsating tandem microbubble for localized and directional single-cell membrane poration.</a> Physical Review Letters. 105, 078101 (2010).</li></ol>

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 ...

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 transfection methods include precipitation with calcium phosphate3, bacterial exotoxins4 and lipofection5. These techniques have proven to be effective; however, certain problems arise such as cell toxicity and non-specificity. Furthermore, techniques such as calcium phosphate precipitation have been found to have transfection efficiencies up to 70% but only in certain cell lines, rarely in primary cells6. This is of note as increasing research efforts are being put into primary cells, especially in the case of designing various treatments for clinical use and the study of DNA functioning.
Temporal disruption of the cell membrane through mechanical stimuli is an alternative for introducing foreign molecules into cells. Techniques include: microinjection of molecules7, electroporation using an electric field to disrupt the membrane8,9, sonoporation using ultrasound waves to disrupt the cell membrane10-12, particle bombardment as demonstrated by the &#34;gene gun&#34;, which shoots particles bound with genes into the cell13, and more recently, the application of fluid shear stress that has been shown to temporally permeabilize mammalian cells14,15. Although mechanical methods avoid some of the aforementioned issues, they are typically accompanied by lower efficiencies and very complex and specialized setups. An atmospheric pressure glow discharge torch (APGD-t) was developed at McGill with the initial goal of functionalizing surfaces and detaching adherent cells in vitro16. Serendipitously, it was discovered that a live/dead stain being used was somehow being taken in by cells without a permeabilizing agent being added. Furthermore, this seemed to have occurred only in restricted areas of the wells which happened to match up with the torch path. Investigation into the permeabilization capabilities of the APGD-t continued and in control studies, it was found that the carrier inert gas jet of the plasma without excitation was also able to permeabilize cells. This contradicted the initial hypothesis that the reactive species created by the plasma jet temporarily impaired the membrane function and suggested that simply the mechanical forces were sufficient to cause cell permeabilization. From here, studies continued in our lab to try and quantify and characterize the permeabilization efficiency of an inert gas jet as well as look at its transfection capabilities16.
Through these studies, it has been found that micropores do in fact form in the plasma membrane, and these pores tend to reseal within approximately 5 sec15. We have demonstrated the technique&#39;s utility in vitro and in vivo in the chorioallantoic membrane of a chick embryo.

<table border="1">
	<tbody>
		<tr>
			<td>Vitality hrGFPII-1</td>
			<td>Agilent Technologies Canada</td>
			<td>240143-57</td>
			<td>Green fluorescent protein for transfection experiments on HeLa cells</td>
		</tr>
		<tr>
			<td>Dextran, AlexaFluor 488; 10,000 MW, Anionic, Fixable</td>
			<td>Life Technologies Inc.</td>
			<td>D22910</td>
			<td>dextran for permeabilization experiments</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Life Technologies Inc.</td>
			<td>L3224</td>
			<td>for counterstaining of mammalian cells</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>for mounting slides when imaging</td>
		</tr>
		<tr>
			<td>Ultra High Purity Helium</td>
			<td>Praxair Canada Inc.</td>
			<td>HE 5.0UH-T</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone DMEM/ High Glucose Media - 500 ml</td>
			<td>Thermo Scientific</td>
			<td>SH30022.01</td>
			<td>for HeLa cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) 1x</td>
			<td></td>
			<td></td>
			<td>Produced in lab</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. List of required reagents</td>
		</tr>
		<tr>
			<td>6-Well Clear TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>#1 22 x 22 Coverslips</td>
			<td>Fisher</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td>WPI Sizing Information</td>
		</tr>
		<tr>
			<td>ID (mm): 1, 0.86, 0.68, 0.5 Order #: 1B200, 1B150, 1B120, 1B100</td>
		</tr>
		<tr>
			<td>Mass-Flo Controller</td>
			<td>MKS</td>
			<td>M100B01314CS1BV</td>
			<td>Mass flow controller, Series M100B, requires an external 12 V power supply</td>
		</tr>
		<tr>
			<td>USB-6009 DAQ Device</td>
			<td>National Instruments</td>
			<td>779026-01</td>
			<td></td>
		</tr>
		<tr>
			<td>UniSlide Assembly with stepping motor and controller</td>
			<td>Velmex</td>
			<td></td>
			<td>Two series MA15 UniSlide Assemblies, fitted with Vexta 1.8&#176; stepping motors provided by Velmex, and controlled by a VXM Stepping Motor Controller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. List of required equipment</td>
		</tr>
	</tbody>
</table>

permeabilization of adhered cells using an inert gas jet

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.

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 &#956;l/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...

Watch this Scientific Journal Video about Permeabilization of Adhered Cells Using an Inert Gas Jet at JoVE.com

Permeabilization of Adhered Cells Using an Inert Gas Jet

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.

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes ...

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