Name: optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells
Uploaded: 2016-07-22T14:00:00
Description: Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

We thank the Life Alliance Organ Recovery Agency of the University of Miami for providing lungs. We also thank Dr. Lisa K&#252;nzi for the DNA preparation used for the experiments shown in Figure 2B and 3-D, Dr. Ben Gerovac and Lisa Novak for the mCherry virus used for the experiments in Figures 3A to C. We also thank Gabriel Gaidosh from the imaging facility, Department of Ophthalmology at the University of Miami.
This study has been sponsored by grants from the NIH, the CF Foundation and the Flight Attendant Medical Research Institute to Dr. Matthias Salathe.

1. Stage 1: Culture of HEK 293T
<ol>
	<li>Prepare HEK cells media (referred as HEK media) by adding 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin to high glucose Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM).</li>
	<li>Coat one 10 cm tissue culture dish with collagen I. Mix 30 &#181;l of collagen I in 2 ml of distilled water. Spread the mix onto the dish and incubate for 2 hr at 37 &#176;C and 5% CO2.</li>
	<li>Remove the mix and let the dish dry in a laminar flow cabinet for 15...

<ol>
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The protocol outlined here assures close to 100% transduction efficiencies. However, there are steps during the process that are important to achieve this goal. For instance, during the transfection process, the presence of precipitates in the transduction mix (drop) or in the dish the day after transduction (Figures 1b and d) are both relevant for a good transfection. The absence of a precipitate or the presence of agglomerates can be due to an omission of one of the transfection reagen...

The development of replication-defective, pseudotyped lentiviruses (LV) has provided researchers with a safe and efficient method to deliver transgenes in vitro 1. Due to their long-term expression, these LV could also be used for the delivery of therapeutic agents in vivo 2,3. The production of LV requires co-transfection of human embryonic kidney (HEK) cells with several plasmids: transfer vector, packaging gag/pol, packaging rev, and envelope vesicular stomatitis virus glycoprotein (VSV-G) plasmids. Third-generation packaging systems are considered safer than second-generation systems because the rev gene is encoded on a separate packaging plasmid, thereby reducing the possibility of recombination to produce a replication competent lentivirus. Although second generation packaging systems yield five fold higher total transduction units, the split of the original viral genome to create the third generation system has decreased the level of transduction only minimally 3,4.
While cell lines can be transduced more easily and efficiently, researchers have shifted their focus to primary cells, because these are more representative of in vivo tissues 5,6. However, primary cells are refractory to transduction. While transduction of fully differentiated airway epithelial cells have been reported, the overall efficiency has not exceeded 15% 7.
Described here is a protocol that allows production of high-titer LVs. A step-by-step approach is outlined to achieve almost 100% transduction efficiency into primary HBE cells. More specifically, the protocol describes the optimal culture conditions instrumental to succeed 3. Briefly, HEK 293T cells are transfected using the lentiviral expression vector pCDH with the EF-1 promoter driving expression of enhanced green fluorescent protein (eGFP) or mCherry, a red fluorescent protein, and a third-generation packaging system. LVs released into the media are collected 24 to 72 hr later. The virus particles are concentrated with polyethylene glycol (PEG), a convenient and easy method of viral concentration, before titer estimation using a p24 antigen enzyme-linked immunosorbent assay (ELISA) kit. Subsequently, undifferentiated primary HBE cells are transduced in proliferation media (bronchial epithelial growth medium or BEGM) 8, while attaching (after being trypsinized), at a multiplicity of infection (MOI) factor of 4, adding hexadimethrine bromide and incubating for 16 hr (overnight). The cells are then allowed to differentiate. Since the viral transgene is expressed long-term (using the appropriate promoter, see discussion), expression will be maintained throughout differentiation into a pseudostratified airway epithelium or can be induced (using an inducible promoter) after differentiation.
This protocol is of broad interest to researchers who want to use primary HBE cells instead of cell lines, and might be adaptable to other difficult to transduce cell types.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEK293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Sigma</td>
			<td>12306C</td>
			<td>use at 10%</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x)</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD Biosciences</td>
			<td>354231</td>
			<td>dilute 1:75 in distilled water</td>
		</tr>
		<tr>
			<td>Calphos</td>
			<td>Clontech</td>
			<td>631312</td>
			<td>Transfection kit containing 2M Calcium solution, 2X HEPES-Buffered solution (HBS) and sterile H2O</td>
		</tr>
		<tr>
			<td>Polythylene glycol 8000</td>
			<td>VWR scientific</td>
			<td>101108-210</td>
			<td>stock of 40%</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A9528</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell 12mm</td>
			<td>Corning</td>
			<td>3460</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C7521</td>
			<td>dilute 1:10 in distilled water</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>stock of 2mg/ml</td>
		</tr>
		<tr>
			<td>Puromycin (10.000x)</td>
			<td>Thermo Scientific</td>
			<td>A11138-03</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Alliance HIV-1 p24 ELISA</td>
			<td>PerkinElmer</td>
			<td>NEK050001KT</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Thermo Scientific</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>pCDH-EF1-MCS-IRES-Puro</td>
			<td>System Biosciences</td>
			<td>CD532A-2</td>
			<td>lentiviral expression vector</td>
		</tr>
		<tr>
			<td>pMD2-VSVG</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pRSV-rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td>packaging DNA</td>
		</tr>
	</tbody>
</table>

optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Figure 1 depicts different key steps of assessing cells and solutions during the transfection process. Figure 1a shows the optimal confluency of HEK293T cells prior to transfection for virus production. It is important that the cells are distributed evenly throughout the dish. Figure 1b reveals the precipitate in a drop of the transfection mixture using bright field optics and a 10X objective. The more the precipitates look like sand grai...

Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

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